![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors: Indirect and Direct Effects on Desensitization
Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science University, Portland, Oregon
Received March 24, 2003; accepted April 16, 2003
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors
was examined in outside-out patches and at glutamatergic synapses in neurons
of the chick cochlear nucleus. A combination of rapid-flow analysis, using
glutamate as an agonist, and kinetic modeling indicated that aniracetam slows
both the rate of channel closing, and the microscopic rates of
desensitization, even for partially liganded receptors. Little effect was
observed on the rate of recovery from desensitization or on the response to
the weakly desensitizing agonist kainate. Aniracetam's effects on receptor
deactivation saturated at lower concentrations than its effects on
desensitization, suggesting that cooperativity between homologous binding
sites was required to regulate desensitization. Analysis of responses to
paired pulses of agonist also indicated that AMPA receptors must desensitize
partially even after agonist exposures too brief to permit rebinding. In the
presence of aniracetam, evoked excitatory synaptic currents (EPSCs) and
miniature EPSCs in low quantal-content conditions had decay times similar to
the time course of receptor deactivation. Under these conditions, the time
course of both transmitter release and clearance must be <1 to 2 ms.
However, in high quantal-content conditions, the evoked EPSC in aniracetam
decayed with a time course intermediate between deactivation and
desensitization, suggesting that the time course of transmitter clearance is
prolonged because of pooling of transmitter in the synaptic cleft. Moreover,
by comparing the amounts of paired-pulse synaptic depression and patch
desensitization prevented by aniracetam, we conclude that significant
desensitization occurs in response to rebinding of transmitter to the AMPA
receptors.
-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors
throughout the brain (e.g., Isaacson and
Nicoll, 1991
; Tang et al.,
1991; Vyklicky et al.,
1991). Studies using rapid flow analysis in neurons
(Vyklicky et al., 1991) or
expressed glutamate receptor 1 (Partin et
al., 1996) suggested that the primary action of the compound is to
slow the rate of channel closure. Such an effect could account both for the
slowing of receptor deactivation and for slowed desensitization, because by
buffering channels in an open configuration, the drug will delay their
accumulation in a desensitized state. Here, drugs may alter macroscopic
desensitization indirectly without interacting specifically with structural
elements involved in the desensitization itself. An alternative hypothesis is
that aniracetam promotes reopening by slowing both desensitization and
unbinding, without effects on channel closure per se
(Arai et al., 1996). This is
suggested by cases in which drugs potentiate glutamate responses and remove
desensitization, with minimal effect on deactivation (Arai et al.,
1996,
2000). However, neither of
these interpretations accounts for a major feature of aniracetam: it blocks
paired pulse depression of synaptic transmission. Finally, interpretation of
aniracetam's effects must account for that fact that functional AMPA receptors
may be composed of two dimers (Sun et al.,
2002). Thus, one would expect multiple binding sites for
allosteric modulators such as aniracetam, and the effects of the drug might
depend on the degree of ligation. It is important, therefore, to develop a
conceptual framework that can account both for the normal behavior of the
receptor and for the targeting of drug action to distinct functions of the
receptor.
In this study, we explored the effects of aniracetam on AMPA receptors in
neurons of the chick cochlear nucleus magnocellularis. Somatic excitatory
synapses cover nearly 50% of the somatic surface area in nucleus
magnocellularis (Parks, 1981).
The accessibility of these receptors to patch-clamp analysis presents an
unusual opportunity for a kinetic analysis of synaptic receptors to accompany
analysis of synaptic transmission. We find that aniracetam slows receptor
deactivation, desensitization, and synaptic currents and reduces paired-pulse
depression of the EPSC. Furthermore, aniracetam may interact with distinct
functional processes of the receptor in a concentration-dependent manner.
Finally, our data confirm that receptor desensitization is strongly dependent
on the quantal content of release, as at high release levels glutamate
clearance is markedly delayed.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
).
The neurons were plated onto poly-D-lysine–coated glass
coverslips and allowed to adhere for 20 min. Isolated neurons were visualized
with a Nikon inverted microscope using phase contrast at 40x.
Intracellular solution contained 70 mM Cs2SO4, 85 mM
sucrose, 4 mM NaCl, 1 mM MgCl2, 10 mM HEPES, and 5 mM BAPTA,
buffered to pH 7.3 with CsOH and osmolarity adjusted to 285 to 295 mOsM. For
rapid application of 10 mM glutamate to patches, a 
-shaped pipette
attached to a piezoelectric translator was used as described previously
(Raman et al., 1994) except
that solution delivery through the -shaped pipette was driven by
nitrogen pressure. Control solutions contained normal Ringer's solution with
200 µM DL-aminophosphonovaleric acid and 10 µM
7-chlorokynurenic acid. Agonist solutions contained the above plus 10 mM
glutamate. The agonist-containing solution was diluted by 5% with
H2O. The patch was blown out after the recording and the solution
exchange time was then measured using the junction potential. The 10 to 90%
solution exchange time was <250 µs. In some experiments, the contents of
the -shaped pipette were changed to normal Ringer's solution and
Ringer's solution diluted with H2O to 10% yielding larger junction
potentials and a more accurate measurement of solution exchange time. The
contents of the -shaped pipette were switched manually with the use of
stopcocks and occurred with a time constant of about 5 s. Data were Bessel
filtered at 10 kHz and digitized at 20 to 40 kHz with pClamp6 software (Axon
Instruments). We have previously shown that the kinetics of AMPA receptors in
acutely dissociated preparations are unaffected by enzymatic treatment and are
kinetically indistinguishable from those in brain slices
(Lawrence and Trussell,
2000).
Curve Fitting and Simulations. Analysis and curve-fitting was
performed with Origin (OriginLab Corp, Northampton, MA), Clamp-fit (Axon
Instruments, Union City, CA), and Igor (Wavemetrics, Lake Oswego, OR). Data
are compared with the results of a kinetic model shown below. A more complex
exponential fit was accepted only if at least a 2-fold improvement in the
2 value was observed. Unless stated otherwise, results are
presented as mean ± S.E.
Simulations were performed with SCoP software (Simulation Resources). The
kinetic model shown in Scheme 1
was employed to interpret the results. Rates are listed in
Table 1. In simulations,
Kf, Kb', and
Kb'' were doubled to account for higher probability
of association and dissociation of two binding sites. This cyclic model, the
conceptual foundation for more complex AMPA/kainate receptor gating schemes
(Raman and Trussell, 1992,
1995b;
Partin et al., 1996;
Hausser and Roth, 1997;
Heckmann and Dudel, 1997),
accounts for several key features of the receptor, including multiple ligand
binding sites and desensitization from partially liganded closed states (the
C1-D1 transition). In the range of glutamate concentrations likely to occur
during synaptic transmission, this minimal model reproduces most major kinetic
features of the AMPA receptor that have been observed experimentally, such as
rates of desensitization and deactivation, paired-pulse desensitization, and
resensitization.
|
|
Slice Recordings. Brainstem slices (250–300 µm) were
prepared from embryonic day 18 chick embryos in an oxygenated saline
containing 140 mM NaCl, 20 mM glucose, 10 mM HEPES, 5 mM KCl, 3 mM
CaCl2, and 1 mM MgCl2, pH 7.35. During recordings,
slices were perfused with room temperature saline at 2 to 4 ml/min in a 1-ml
chamber. Neurons were viewed with a Zeiss Axioskop and Olympus 60x water
immersion lens using Nomarski optics and infrared illumination. For
measurement of EPSCs, saline was supplemented with 100 µM
DL-aminophosphonovaleric acid, 10 µM 7-Cl-kynurenate, 10 µM
SR-95531, and 2 µM strychnine. Neurons in brain slices were voltage-clamped
with an Axopatch 200B amplifier (Axon Instruments). Electrode series
resistances (2–8 M
) were compensated 80 to 95%. Pipettes were
filled with an intracellular solution containing 125 mM Cs-methanesulfonate,
15 mM CsCl, 10 mM HEPES, 5 mM BAPTA, and 1 mM MgCl2, pH 7.25.
Spontaneous mEPSCs were obtained at a holding potential of -60 mV. Currents
were filtered at 10 kHz and sampled at 50 kHz. Events were detected using a
template detection algorithm implemented in Axograph software (Axon
Instruments). Evoked synaptic responses in neurons clamped to -30 mV were
obtained by positioning a stimulus electrode (1.5–3 M) onto
nearby myelinated fibers, and individual auditory nerve axons stimulated by
100 to 200 µs, 5 to 50 V pulses delivered by an isolated stimulus unit
(AMPI Iso-flex). Currents were filtered at 5 to 10 kHz and sampled at 20 to 50
kHz. Aniracetam stocks (0.5 M, 100x) were prepared in DMSO and added to
extracellular solutions immediately before use. The final working
concentration of aniracetam was 5 mM, and aniracetam-containing solutions
included 1% (v/v) DMSO. For all experiments using aniracetam, control
extracellular solutions were also supplemented with 1% DMSO. Chemicals and
drugs were obtained from Sigma (St. Louis, MO), RBI/Sigma (Natick, MA), and
Tocris Cookson (Ballwin, MO).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Lawrence and
Trussell, 2000). Figure
1A illustrates a control response and exponential fits to the
onset of desensitization. The inset displays the residuals of the single and
double exponential fits, showing that the latter provided the best description
of the decay phase of the current (1 exponential 2 = 11.6
± 7.8; two-exponential 2 = 0.50 ± 0.15; 23-fold
improvement; see Materials and Methods).
Table 2 summarizes the average
exponential components obtained for these fits. As shown in
Fig. 2D, receptors desensitized
by 98.9 ± 0.3% (n = 6). With increase in the concentration of
aniracetam, slower exponential components of desensitization became dominant;
initially, a 7-ms component became dominant
(Fig. 1B), and at the highest
aniracetam concentrations, an additional component of about 30 ms was
prominent (Fig. 1C;
Table 2). At an intermediate
concentration of aniracetam (1 mM), three exponentials were required to fit
the entire decay phase, and included the 1-, 7-, and 30-ms components
(Fig. 1B,
Table 2). At 5 mM, the extent
of desensitization, estimated by the extrapolation of the exponential fits to
infinite time, was approximately 90.2 ± 1.5%
(Fig. 1D).
|
|
|
The deactivation of currents after the rapid removal of glutamate without
aniracetam was generally described with a single exponential decay of 500 to
600 µs (Fig. 1D), consistent
with previous observations (Raman and Trussell,
1992,
1995b). In the presence of as
little as 0.1 mM aniracetam, the time constant of deactivation doubled,
doubling yet again at 5 mM (Figs.
1B,
2A, and C). Over the full range
of concentrations, deactivation increased from 0.55 ± 0.19 ms
(n = 5) in control to 1.95 ± 0.52 ms (n = 8) with 5
mM aniracetam (Figs. 1F and
2A). Differences in time
constant between 1 and 5 mM were not significant (p = 0.24). In
simulations (see Discussion), it was possible to describe the
relationship between deactivation and aniracetam concentration as a steady
decrease in , the rate constant of channel closing
(Fig. 2A, dashed line).
Although it was not possible to know whether the effects of aniracetam
saturated because of drug solubility limits, the majority of its effects on
deactivation appeared at 0.1 to 1 mM. We did not consistently observe multiple
exponential decays in deactivation.
The magnitude of the effects of aniracetam on desensitization seemed to be
larger and occurred at a higher aniracetam concentration than the effects on
deactivation. Figure 2B
illustrates the results of weighted exponential fits to the onset of
desensitization, which provide a convenient index of the overall slowing of
the decaying current. The time constant in controls is between 1 and 2 ms
(Fig. 2B), doubles in 0.1 mM
aniracetam, but is more than 5- and 10-fold longer in 1 and 5 mM aniracetam
(Fig. 2C), respectively.
Examining the maximal effect of the drug, desensitization slowed from about
1.7 ± 0.1 ms (n = 6) in control to 21.2 ± 1.7 ms
(n = 10) in 5 mM aniracetam. Using the same model parameters for
Fig. 2A, the slowing of
desensitization expected for an action solely on is shown in
Fig. 2B as a dashed line.
Clearly, values of suitable to explain deactivation are not sufficient
to account for aniracetam's effects on desensitization. This point is
emphasized in Fig. 2C, which
plots the dose dependence of the -fold increase in time constants for patches
and for the model. Beginning at 1 mM aniracetam, and model parameters suitable
to account for deactivation, a sharp deviation from the model predictions were
observed for desensitization. Moreover, an effect solely on was not
adequate to account for the increase in steady-state current during long
glutamate applications in 3 and 5 mM aniracetam
(Fig. 2D). These data indicate
that at high concentrations, aniracetam must act on the microscopic rates of
entry into desensitized states, in addition to slowing channel closure.
Paired-Pulse Desensitization and Resensitization. When pairs of
brief pulses of glutamate are delivered, receptors continue to desensitize
between the two applications, despite the absence of free glutamate
(Raman and Trussell, 1995b).
If a modulator reduced desensitization to brief conditioning pulses, then it
must be acting directly on processes unrelated to the rate of opening or
closing of the channel, because the channels are closed during the period
between the two pulses. Figure 3A,
i, illustrates a protocol in which two pulses of 10 mM glutamate
were applied to a patch, with the pulses separated by 10 ms and the duration
of the first pulse (P1) increased with each repetition of the pair. Responses
to P1 reached the same peak even for the briefest applications (compare
shortest and longest junction potentials in
Fig. 3A, i). For such brief
conditioning pulses (
0.45 ms), the second pulse (P2) produced a response
42% smaller than the first (Fig. 3A,
ii). As P1 was increased, the response to P2 grew progressively
smaller, because of greater desensitization, until a maximum of 62% reduction
in the peak at the longest P1 (3.45 ms;
Fig. 3A, iii).
Figure 3B, i, illustrates the
prediction that paired-pulse desensitization should be resistant to modulators
that act only on the channel-closing rate by showing the relative amount
amounts of paired-pulse desensitization with brief
(Fig. 3B, ii) or long
(Fig. 3B, iii) conditioning
pulses when the closing rate is changed. Here, the amount of desensitization
after a very brief pulse is expected not to change when the closing rate is
reduced. Thus, a paired-pulse protocol, measuring the amount of
desensitization and its rate of recovery, can be used to determine whether
modulators alter particular desensitization steps independent of effects on
channel closing rates.
|
However, in the presence of 3 or 5 mM aniracetam, much less desensitization
was apparent even for the briefest initial application
(Fig. 3C, i–iii).
Figure 3E illustrates data
pooled from four patches in which both control (
) and aniracetam (
)
was obtained with a wide range of pulse durations. In each patch, the
amplitude of P2 was normalized to the amplitude of P1, which corrects for
variability among patches and the effects of aniracetam on peak current (see
below). The duration of each initial pulse was slightly variable, especially
for the briefest pulses, and thus we have plotted the normalized P2 responses
against the half-width of the P1 application (see Materials and
Methods). For short conditioning pulses (mean 0.6 ± 0.1 ms), the
average response to P2 was 0.56 ± 0.02 (n = 5) after
normalizing to the peak amplitude of the P1 response. For a long conditioning
pulse (3.1 ± 0.03 ms), the average was 0.37 ± 0.03. In these
same patches, aniracetam increased the amplitude of the response to P1 by 31
± 13% (3 mM, n = 8) and 37 ± 31% (5 mM, n =
7). For the short pulses (0.6 ± 0.05 ms), the normalized response to P2
was 0.84 ± 0.02 (n = 5), not significantly different from that
of the response to long (3.0 ± 0.2 ms) pulses, whose mean normalized
amplitude was 0.78 ± 0.02 (p = 0.067, unpaired t
test).
These data indicate, first, that desensitization was observed even with the briefest glutamate pulses. Solid exponential curves through the data in Fig. 3E were extrapolated to zero time to estimate the minimal amount of desensitization the receptors would be expected to undergo. These curves intersect the zero time axis at 0.64 for control and 0.86 for aniracetam (Fig. 3D). Thus, in control, a minimum of 36% of receptors must desensitize. Even with aniracetam, some desensitization (14%) was obtained with minimal exposure to glutamate.
A second implication of these observations was apparent upon simulations of
this experiment using the model discussed below. In
Fig. 3E, the solid thick line
through the open circles shows the predictions of the kinetic model with the
control parameter set. The coincidence of the model and the control data
indicate that the model can account for the paired pulse responses reasonably
well. When the value of was changed to account for deactivation in 5
mM aniracetam, the resulting simulation was unable to approximate the
experimentally obtained aniracetam data
(Fig. 3E, dashed line).
Instead, additional modifications were made in two desensitization rates,
Lf and Mf, which were reduced 5- and 4.2-fold,
respectively. The resulting simulations
(Fig. 3E, thick line through
open squares) were successful in describing the experimental effects of
aniracetam. Thus, these data support the conclusion from
Fig. 2 that aniracetam must
directly lower the microscopic rates of desensitization.
The paired-pulse desensitization results described above suggested that
rates coupled directly to desensitized states may be affected by aniracetam.
To determine whether microscopic rates of recovery from desensitization
(Lb and/or Mb) are sensitive to aniracetam, we measured
recovery after desensitizing pulses of glutamate. In the absence of
aniracetam, the recovery rates induced by either a brief or long pulse have
been shown to be similar, occurring in an exponential time course of
approximately 16 ms (Raman and Trussell,
1995b). In the present study, accumulation into desensitized
states was driven by a 20-ms conditioning pulse of glutamate and recovery from
desensitization was monitored with 1-ms test pulses at intervals lengthened in
3-ms increments. Example records are shown in
Fig. 4A. The amplitude of the
test pulse was divided by the amplitude of the first pulse in
Fig. 4C. The data were fitted
by single exponential of 19 ± 2 ms (n = 3), similar to the
value of 16 ms reported previously for these glutamate receptors
(Raman and Trussell, 1995b). A
short lag in recovery was accounted for by a shift in the time axis of
exponential fits, as described previously
(Raman and Trussell, 1995b).
Recovery from desensitization was then measured in the presence of 5 mM
aniracetam using an identical conditioning/test pulse protocol
(Fig. 4B), and averaged
recovery data are plotted in Fig.
4C. Data were fit with a single exponential of 16 ± 1 ms
(n = 8), indicating that the recovery time course of desensitization
was not markedly altered by aniracetam (unpaired t test, p =
0.17). Thus, coupled with evidence that rates directly associated with
desensitization must be influenced to account for both the reduction of
paired-pulse desensitization and slowing of the rate of desensitization by
aniracetam, we suggest that Lf and/or Mf are slowed by
aniracetam. Accordingly, simulations using the parameter set described above
gave exponential recovery times of 20.3 and 15.6 ms for the control and
aniracetam models, respectively.
|
The measurement of recovery time course gave us the opportunity to test the
validity of the model with respect to the observations that recovery proceeds
after a delay. This observation (Raman and
Trussell, 1995b; Bowie and
Lange, 2002; Robert and Howe,
2003) has been taken as evidence of conformational steps preceding
the full recovery of desensitized receptors, a feature predicted from recent
allosteric models of AMPA receptor gating
(Bowie and Lange, 2002;
Robert and Howe, 2003).
Figure 4D shows that our
earlier model (dashed line) also predicts this delay in recovery quite well,
deviating markedly from a simple exponential increase in sensitivity (solid
black line). This observation indicates that the model is indeed useful for
evaluating alterations in receptor function that could account for the action
of modulators of desensitization.
Aniracetam Increases Channel Open Probability. Nonstationary
variance analysis was employed to estimate the open probability of the AMPA
receptor in the presence of 5 mM aniracetam. Patches were exposed at least 30
times to 100-ms pulses of glutamate. The current variance was plotted against
the mean current and fitted with a parabola in the form 
2
(t) = i x I(t) -
I2(t)/N, where 2 (t)
is the variance, I2(t) is the mean, i is
the single channel current, and N is the number of receptors in the
patch (not shown). The peak open probability (Po) can be calculated
from Imax/iN, where Imax is
the maximal current and I and N are from the fit. In three
patches from which stable responses were obtained without appreciable run-down
of peak current, the Po was 0.72 ± 0.01, i was 1.9
± 0.2 [which corresponds to a single-channel conductance (
) of
26 ± 3 pS], and N was 196 ± 70 channels. These results indicate
a higher Po and from that previously observed in the
presence of 10 mM glutamate alone, in which Po was 0.54 ±
0.1 and was 18 ± 4 pS
(Raman and Trussell, 1995a).
For comparison, the model developed here yielded a peak Po of 0.57
and 0.83 for control and aniracetam.
Aniracetam Does Not Prolong Deactivation in Kainate. The agonist
kainate acts on AMPA receptors, generating a current that produces little
desensitization but instead rapidly attains a large, steady-state level
(Raman and Trussell, 1992).
Thus, with kainate, one would expect the AMPA receptor to spend less time in
desensitized states (Patneau and Mayer,
1991). However, deactivation of the kainate-induced response, as
with glutamate, should still be dependent on the channel-closing rate .
We therefore explored the effect of aniracetam on deactivation in kainate. In
contrast to aniracetam's effect on glutamate currents, we found that
aniracetam did not significantly affect deactivation of kainate currents
(Fig. 5, B and E). Furthermore,
neither peak amplitude (Fig.
5C) nor desensitization time constants
(Fig. 5, A and E) were
affected. However, a small but significant enhancement of steady-state current
was detected (Fig. 5D). Thus,
the value of , as well as its sensitivity to aniracetam, seems to
depend on the type of agonist. Shen et al.
(1999) also observed little
effect of aniracetam on current evoked by kainate.
|
Kinetics of Synaptic Transmission. Excitatory synaptic currents were
recorded in nucleus magnocellularis neurons in brain slices to determine how
their decay time compares with that of glutamate responses in patches. The
sensitivity of these currents to AMPA receptor antagonists, to cyclothiazide,
aniracetam, and GYKI 52466 (Trussell et
al., 1993; Brenowitz and
Trussell, 2001) strongly suggests that they are mediated by the
same receptors observed in outside-out patches. In control solutions, channel
deactivation and desensitization may be similar in time course to that of
transmitter diffusion, complicating comparisons of EPSCs to patch responses.
Here, we focused on the responses in the presence of 5 mM aniracetam at room
temperature; in this condition, transmitter diffusion after release of single
glutamate vesicles should be considerably faster than channel kinetics, so
interpretable comparisons between patch and synaptic responses could be made.
Figure 6A and
Table 3 summarize measurements
of kinetics and amplitudes of mEPSCs in control and aniracetam. In addition,
we compared the decay time of mEPSCs to deactivating responses in patches, all
with aniracetam (Table 3). For
cells in which mEPSCs were best fit with a sum of two exponentials, both
components increased in aniracetam. The single-exponential decay constants of
mEPSCs and deactivating responses in patches were not significantly different.
In addition, the increase in amplitude of mEPSCs by aniracetam was similar to
the increase observed in patch responses
(Table 3). We conclude that
receptors in patches and at synapses have similar kinetics and sensitivity to
aniracetam. Transmitter clearance times must therefore be considerably faster
than 1.5 ms (the decay time constant of mEPSCs in anirecetam). Indeed, because
mEPSCs and deactivation is similar even in control solutions
(Otis et al., 1996a),
clearance of transmitter from one vesicle must be <0.5 ms.
|
|
During evoked transmission, EPSC decay times are expected to be broader
than mEPSCs, because of temporal dispersion of release and delayed clearance
of transmitter. We measured the decay time of evoked EPSCs (eEPSCs) in the
presence of 10 µM Cd2+ to reduce the quantal content
and so reduce the problems of clearance of transmitter from the calyceal
synaptic cleft. Experiments were performed by recording eEPSCs first in
control solution, then in bath-applied aniracetam, and finally in the presence
of aniracetam plus 10 µM Cd2+, applied by pressure
application from a nearby pipette. In Fig.
6B, amplitudes and kinetics of eEPSCs are shown for all of these
conditions. Aniracetam increased the amplitude of evoked EPSCs to a somewhat
larger degree than in mEPSCs and patches
(Table 3). Evoked EPSCs rose
more slowly after application of aniracetam in low (0.70 ± 0.07 ms) or
high (0.83 ± 0.10 ms) release conditions (control 0.45 ± 0.02
ms, n = 4). Decay times could be best fit with two exponentials. In
aniracetam plus Cd2+, these components were similar to
the double exponential decay of mEPSCs in aniracetam
(Table 3). For single
exponential fits, the decays were also similar (2 and 1.5 ms for eEPSCs and
mEPSCs, respectively). These data suggest 1) that aniracetam itself does not
cause a severe distortion of the release time course, as has been suggested
for cyclothiazide (Diamond and Jahr,
1995), and 2), that the clearance of transmitter is probably
faster than the deactivation time of 1.5 to 2 ms.
In solutions in which release was not blocked, eEPSCs decayed much more
slowly, as shown in Fig. 6B and
Table 3. These values were more
than 2-fold longer than the time constants observed in
Cd2+ or for mEPSCs. Because aniracetam did not markedly
prolong the release process itself, the slower decay in high-release
conditions is most likely to be an outcome of two factors: slower transmitter
diffusion will lead to rebinding and reactivation of receptors, so that the
decay would reflect the gradual decline in glutamate levels. However,
rebinding will also lead to desensitization, and the kinetics of decay could
in part reflect this process. Table
3 summarizes the rates of decay of synaptic events, deactivation
in aniracetam, and can be compared with values for desensitization in
Table 2. In 5 mM aniracetam,
desensitization to step pulses of glutamate occurs with time constants of 7
and 28 ms, the latter being the main component of the fit. eEPSCs in
aniracetam decay with a time course that is generally faster and suggests that
diffusion/rebinding is the dominant factor in shaping the eEPSC under these
conditions. More importantly, the data strongly suggests that, under
conditions of high release, that glutamate clearance is markedly slower than
the time course of desensitization of AMPA receptors in the absence
of aniracetam (1 ms; Raman and
Trussell, 1995b).
To compare depression of EPSCs to the paired-pulse desensitization
described in Fig. 3, we next
examined the response to pairs of synaptic stimuli, delivered at 10-ms
intervals above. Figure 7, A and
B, show that in aniracetam, depression was markedly lessened, as
described previously (Brenowitz and
Trussell, 2001). Depression arises both from desensitization and
from presynaptic effects, such as depletion of transmitter. Assuming no
presynaptic effects of aniracetam
(Brenowitz and Trussell, 2001),
the difference in depression with and without aniracetam should give an
estimate of the magnitude of synaptic desensitization. This value, calculated
as PPDani/PPDcontrol for each cell (PPD is defined as
the ratio of the second to the first response in the pair), gave an average
value of 2.04 ± 0.16 (n = 16) and is plotted in
Fig. 7C as a horizontal solid
line. For comparison, this same parameter is derived from the exponential fits
of data from patches and plotted in Fig.
7C against the interval between pulses (continuous curved line).
show the predictions of the model derived above, which agreed well with
the data. show the predictions for a model in which aniracetam only
slows . With the shortest glutamate pulses, the ratio was about 1.5,
increasing to about 2 with the longest pulses. The ratios for synaptic and
patch data cross at a P1 of around 4 ms. Thus, the degree of desensitization
10 ms after transmitter release is equivalent to that produced by a square
pulse of 10 mM glutamate lasting about 4 ms, with a 10 ms-period of
recovery.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
;
Suppiramaniam et al., 2001).
Consequently, both the rates of deactivation and desensitization were slowed.
Indeed, the slowing of desensitization is clearly linked to the slowing of
deactivation at concentrations of aniracetam <1 mM. Stabilization of the
open conformation would also be expected to increase the peak amplitude of
responses, before the onset of desensitization. However, at higher
concentrations of aniracetam, we observed a further slowing and lessening of
desensitization, beyond what would be predicted from an effect solely on
channel gating but without further slowing of deactivation. Such an action
suggested that binding of additional aniracetam molecules had an effect
directly upon the rates governing entry into desensitized states. This
conclusion was further supported by two observations. First, paired-pulse
desensitization was markedly reduced by aniracetam, an effect not expected
from a slowing of channel closure. Second, the rate of recovery from
desensitization was only slightly faster in aniracetam. All of these effects
could be explained in our model by a dual action of the drug. The fact that
effects on deactivation and additional slowing of desensitization had
different dose dependence suggests that desensitization can be controlled
independently of deactivation. Such a conclusion is consistent with the fact
that different AMPA receptor subunits exhibit similar deactivation rates but
very different desensitization rates
(Mosbacher et al., 1994;
Koike et al., 2000). Thus, in
native receptors, desensitization is a process fundamentally different from
channel closure.
Based on these differences in the dose dependence of deactivation and
desensitization, it seems likely that aniracetam can have a significant effect
with only a fraction of the subunits occupied by the drug. Current models of
AMPA receptor gating suggest that receptors may open to subconductance states
when a fraction of the subunits is bound to glutamate
(Rosenmund et al., 1998;
Smith and Howe, 2000). It
might be expected then that aniracetam slows the gating for a fraction of the
subunits, and this should result in multiphasic decay, with slower components
increasing in amplitude with increase in aniracetam concentration.
Unfortunately, biphasic deactivation was difficult to resolve in each case.
The apparently low affinity of aniracetam also complicates the analysis,
because the drug may unbind and rebind during the course of deactivation.
At higher concentrations of aniracetam, the magnitude of the effects on
deactivation and desensitization diverged sharply, implying that
desensitization is controlled by coordinate properties of multiple subunits.
This theme has been explored by several recent studies examining the
biophysical properties of recombinant AMPA receptors. Robert et al.
(2001), expressed mixtures of
desensitizing and largely nondesensitizing (mutant) subunits and found that
different rates of desensitization were dependent on the stoichiometry of the
mixture. With receptors composed of equal parts of the two subunit types,
receptors desensitized with fast, intermediate, and very slow decay times.
Their interpretation was that the intermediate component arose from receptors
that contained some mutant subunits that conferred both slower deactivation
and desensitization to the receptor. In their view, only when the receptor
contained at least two mutant subunits present as dimers did the receptor lose
most of its desensitization; thus, a critical subunit structure determined
desensitization of the whole receptor. In the present study, increase in
aniracetam concentration produced distinct shifts in the rate of
desensitization reminiscent of the results of Robert et al.
(2001) At low aniracetam
concentrations, the desensitization onset (7 ms 
) was similar to
that predicted by the observed slowing of channel closure. At higher
concentrations, a still slower rate of 20 to 30 ms emerged that eventually
dominated the decay process. Presumably, when a critical number of aniracetam
molecules bind the receptor, a global shift in desensitization is effected
that is distinct from the processes governing the rate of channel closure.
Sun et al. (2002) examined
the properties of recombinant receptors with mutations affecting
desensitization or with the modulator cyclothiazide, contrasting these with
the changes in affinity between pairs of subunit fragments. They concluded
that desensitization is associated with, or caused by, a destabilization of
subunit dimers, and that cyclothiazide acts by binding between subunits and
disturbing the change in dimer affinity. It is not yet clear where aniracetam
binds in the AMPA receptor, and binding between subunits is a possibility. One
issue not addressed in that study, however, was how pairs of dimers, which
form the receptor, interact during the process of desensitization. Together
with the interpretations of Robert et al.
(2001), it may be that failure
of a single dimer to undergo the structural arrangements characteristic of
desensitization will prevent the entire receptor from desensitizing. If, in
our experiments, the full effect of aniracetam was associated with binding at
both dimer pairs, then it may be that interactions between dimer
pairs are part of the process of desensitization as well. However, aniracetam
is a very low-affinity agent, and it remains possible that the effects we
observe reflects multiple binding events within single dimers. This
possibility is compounded by the possible heteromeric structure of auditory
AMPA receptors, containing glutamate receptors 3 and 4 flop, which could
present binding sites of different affinity
(Wang et al., 1998;
Ravindranathan et al., 2000;
Gardner et al., 2001).
Aniracetam As a Tool to Probe Synaptic Transmission. Previous
studies suggested that, in nucleus magnocellularis, transmitter could
desensitize receptors, but it was not clear whether synaptic desensitization
occurred by transmitter rebinding or if it was instead a reflection of that
fraction of receptors that desensitize immediately after binding glutamate
(Otis et al., 1996b). In this
study, this issue was approached first by detailed comparison of EPSCs to the
responses to glutamate in patches, all in the presence of aniracetam. We found
that the deactivation rate was similar to the decay time of mEPSCs or of
evoked EPSCs under conditions of low quantal content. However, these decay
times were markedly slower than that of evoked EPSCs of high quantal content
(i.e., without Cd2+). In the absence of aniracetam,
desensitization is quite rapid ( of 1 ms) and so it is difficult to
infer the relative contributions to the EPSC decay of desensitization,
transmitter diffusion, and transmitter release time course. In the presence of
the drug, however, the EPSC decay was slower than the release time course and
intermediate between deactivation and desensitization. Thus, these data
indicate that transmitter clearance during the unblocked, evoked EPSC is slow,
lasting at least several ms, and therefore must lead to rebinding and
reactivation of receptors. Given the speed of desensitization in control
solutions (Raman and Trussell,
1995b), the data support the idea that some desensitization may
occur by rebinding of transmitter.
Experiments with paired synaptic stimuli support this conclusion. The
extent of protection from depression by aniracetam was similar to that seen
for a glutamate stimulus delivered 10 ms after a 4-ms square pulse of 10 mM
glutamate. Although the actual concentration transient is certainly smaller
and slower than this (Otis et al.,
1996a; Sakaba and Neher,
2001), it should be noted that if clearance were significantly
faster, the effect of aniracetam on depression would have been less: based on
the data on patches, with the briefest exposure to glutamate, aniracetam
should have increased the size of the scaled second EPSC by only 40%. Instead,
the EPSC was doubled, suggesting that the glutamate transient lasts long
enough to cause significant rebinding and additional desensitization.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: AMPA,
-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; EPSC, excitatory
postsynaptic current; BAPTA,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid; SR-95531, 2-(3-carboxyl)-3-amino-6-(4-methoxyphenyl)-pyridazinium
bromide; DMSO, dimethyl sulfoxide; Po, open probability; GYKI
52466, 1-(4-aminophenyl)-methoxy-7,8-methylenedioxy-5H-2,3- benzodiazepine
hydrochloride; mEPSC, miniature excitatory postsynaptic current; eEPSC, evoked
excitatory postsynaptic current; PPD, paired-pulse depression.
1 Present address: Unit on Cellular and Synaptic Physiology, Laboratory of
Cellular and Synaptic Neurophysiology, National Institute of Child Health and
Human Development, National Institutes of Health, Bethesda, Maryland. 
2 Present address: Department of Neurobiology, Harvard Medical School, 220
Longwood Ave, Boston, Massachusetts 02115.
Address correspondence to: Dr. Laurence Trussell, Auditory Neuroscience, L-335A, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd., Portland OR 97239. E-mail: trussell{at}ohsu.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Arai AC, Kessler M, Rogers G, and Lynch G (2000)
Effects of the potent ampakine CX614 on hippocampal and recombinant AMPA
receptors: interactions with cyclothiazide and GYKI 52466. Mol
Pharmacol 58:
802–813.
Bowie D and Lange GD (2002) Functional stoichiometry
of glutamate receptor desensitization. J Neurosci
22:
3392–3403.
Brenowitz S and Trussell LO (2001) Minimizing synaptic
depression by control of release probability. J
Neurosci 21:
1857–1867.
Diamond JS and Jahr CE (1995) Asynchronous release of synaptic vesicles determines the time course of the AMPA receptor-mediated EPSC. Neuron 15: 1097–1107.[CrossRef][Medline]
Gardner SM, Trussell LO, and Oertel D (2001)
Correlation of AMPA receptor subunit composition with synaptic input in the
mammalian cochlear nuclei. J Neurosci
21:
7428–7437.
Hausser M and Roth A (1997) Dendritic and somatic
glutamate receptor channels in rat cerebellar Purkinje cells. J
Physiol 501:
77–95.
Heckmann M and Dudel J (1997) Desensitization and resensitization kinetics of glutamate receptor channels from Drosophila larval muscle. Biophys J 72: 2160–2169.[Medline]
Isaacson JS and Nicoll RA (1991) Aniracetam reduces
glutamate receptor desensitization and slows the decay of fast excitatory
synaptic currents in the hippocampus. Proc Natl Acad Sci
USA 88:
10936–10940.
Koike M, Tsukada S, Tsuzuki K, Kijima H, and Ozawa S
(2000) Regulation of kinetic properties of GluR2 AMPA receptor
channels by alternative splicing. J Neurosci
20:
2166–2174.
Lawrence JJ and Trussell LO (2000) Long-term
specification of AMPA receptor properties after synapse formation.
J Neurosci 20:
4864–4870.
Mosbacher J, Schoepfer R, Monyer H, Burnashev N, Seeburg PH, and
Ruppersberg JP (1994) A molecular determinant for submillisecond
desensitization in glutamate receptors. Science (Wash
DC) 266:
1059–1062.
Otis TS, Wu YC, and Trussell LO (1996a) Delayed
clearance of transmitter and the role of glutamate transporters at synapses
with multiple release sites. J Neurosci
16:
1634–1644.
Otis T, Zhang S, and Trussell LO (1996b) Direct
measurement of AMPA receptor desensitization induced by glutamatergic synaptic
transmission. J Neurosci
16:
7496–7504.
Parks TN (1981) Morphology of axosomatic endings in an avian cochlear nucleus: nucleus magnocellularis of the chicken. J Comp Neurol 203: 425–440.[CrossRef][Medline]
Partin KM, Fleck MW, and Mayer ML (1996) AMPA receptor
flip/flop mutants affecting deactivation, desensitization, and modulation by
cyclothiazide, aniracetam and thiocyanate. J Neurosci
16:
6634–6647.
Patneau DK and Mayer ML (1991) Kinetic analysis of interactions between kainate and AMPA: evidence for activation of a single receptor in mouse hippocampal neurons. Neuron 6: 785–798.[CrossRef][Medline]
Raman IM and Trussell LO (1992) The kinetics of the response to glutamate and kainate in neurons of the avian cochlear nucleus. Neuron 9: 173–186.[CrossRef][Medline]
Raman IM and Trussell LO (1995a) Concentration-jump analysis of voltage-dependent conductances activated by glutamate and kainate in neurons of the avian cochlear nucleus. Biophys J 69: 1868–1879.[Medline]
Raman IM and Trussell LO (1995b) The mechanism of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor desensitization after removal of glutamate. Biophys J 68: 137–146.[Medline]
Raman IM, Zhang S, and Trussell LO (1994) Pathway-specific variants of AMPA receptors and their contribution to neuronal signaling. J Neurosci 14: 4998–5010.[Abstract]
Ravindranathan A, Donevan SD, Sugden SG, Greig A, Rao M, and Parks
TN (2000) Contrasting molecular composition and channel
properties of AMPA receptors on chick auditory and brainstem motor neurons.
J Physiol 523:
667–684.
Robert A and Howe JR (2003) How AMPA receptor
desensitization depends on receptor occupancy. J
Neurosci 23:
847–858.
Robert A, Irizarry SN, Hughes TE, and Howe JR (2001)
Subunit interactions and AMPA receptor desensitization. J
Neurosci 21:
5574–5586.
Rosenmund C, Stern-Bach Y, and Stevens CF (1998) The
tetrameric structure of a glutamate receptor channel. Science (Wash
DC) 280:
1596–1599.
Sakaba T and Neher E (2001) Quantitative relationship
between transmitter release and calcium current at the calyx of held synapse.
J Neurosci 21:
462–476.
Shen Y, Lu T, and Yang XL (1999) Modulation of desensitization at glutamate receptors in isolated crucian carp horizontal cells by concanavalin A, cyclothiazide, aniracetam and PEPA. Neuroscience 89: 979–990.[CrossRef][Medline]
Smith TC and Howe JR (2000) Concentration-dependent substate behavior of native AMPA receptors. Nat Neurosci 3: 992–997.[CrossRef][Medline]
Sun Y, Olson R, Horning M, Armstrong N, Mayer M, and Gouaux E (2002) Mechanism of glutamate receptor desensitization. Nature (Lond) 417: 245–253.[CrossRef][Medline]
Suppiramaniam V, Bahr BA, Sinnarajah S, Owens K, Rogers G, Yilma S, and Vodyanoy V (2001) Member of the Ampakine class of memory enhancers prolongs the single channel open time of reconstituted AMPA receptors. Synapse 40: 154–158.[CrossRef][Medline]
Tang CM, Shi QY, Katchman A, and Lynch G (1991)
Modulation of the time course of fast EPSCs and glutamate channel kinetics by
aniracetam. Science (Wash DC)
254:
288–290.
Trussell LO, Zhang S, and Raman IM (1993) Desensitization of AMPA receptors upon multi-quantal neurotransmitter release. Neuron 10: 1185–1196.[CrossRef][Medline]
Vyklicky L Jr, Patneau DK, and Mayer ML (1991) Modulation of excitatory synaptic transmission by drugs that reduce desensitization at AMPA/kainate receptors. Neuron 7: 971–984.[CrossRef][Medline]
Wang YX, Wenthold RJ, Ottersen OP, and Petralia RS
(1998) Endbulb synapses in the anteroventral cochlear nucleus
express a specific subset of AMPA-type glutamate receptor subunits.
J Neurosci 18:
1148–1160.
This article has been cited by other articles:
|
|
 |
|
  H. Yang and M. A. Xu-Friedman Relative Roles of Different Mechanisms of Depression at the Mouse Endbulb of Held J Neurophysiol, May 1, 2008; 99(5): 2510 - 2521. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. H. Ahmed, A. P. Loh, D. E. Jane, and R. E. Oswald Dynamics of the S1S2 Glutamate Binding Domain of GluR2 Measured Using 19F NMR Spectroscopy J. Biol. Chem., April 27, 2007; 282(17): 12773 - 12784. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Jin, S. Clark, A. M. Weeks, J. T. Dudman, E. Gouaux, and K. M. Partin Mechanism of Positive Allosteric Modulators Acting on AMPA Receptors J. Neurosci., September 28, 2005; 25(39): 9027 - 9036. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Turetsky, E. Garringer, and D. K. Patneau Stargazin Modulates Native AMPA Receptor Functional Properties by Two Distinct Mechanisms J. Neurosci., August 10, 2005; 25(32): 7438 - 7448. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-F. Xia, M. Kessler, and A. C. Arai Positive {alpha}-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Modulators Have Different Impact on Synaptic Transmission in the Thalamus and Hippocampus J. Pharmacol. Exp. Ther., April 1, 2005; 313(1): 277 - 285. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
2 values display the fold-improvement from single- to
double-exponential fit.
