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Department of Biology, Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02454-9110
Submitted 27 January 2003; accepted in final form 28 March 2003
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ABSTRACT |
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-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptor complements across the cortex, and to investigate NMDA receptor
(NMDAR)-based models of persistent activity, we compared NMDA/AMPA ratios in
prefrontal (PFC) and visual cortex (VC) in rat. Whole cell voltage-clamp
responses were recorded in brain slices from layer 2/3 pyramidal cells of the
medial PFC and VC of rats aged p16–p21. Mixed miniature excitatory
postsynaptic currents (mEPSCs) having AMPA receptor (AMPAR)- and
NMDAR-mediated components were isolated in nominally 0
Mg2+ ACSF. Averaged mEPSCs were well-fit by double
exponentials. No significant differences in the NMDA/AMPA ratio (PFC: 27
± 1%; VC: 28 ± 3%), peak mEPSC amplitude (PFC: 19.1 ± 1
pA; VC: 17.5 ± 0.7 pA), NMDAR decay kinetics (PFC: 69 ± 8 ms;
VC: 67 ± 6 ms), or degree of correlation between NMDAR- and
AMPAR-mediated mEPSC components were found between the areas (PFC: n
= 27; VC: n = 28). Recordings from older rats (p26–29) also
showed no differences. EPSCs were evoked extracellularly in 2 mM
Mg2+ at depolarized potentials; although the average
NMDA/AMPA ratio was larger than that observed for mEPSCs, the ratio was
similar in the two regions. In nominally 0 Mg2+ and in
the presence of CNQX, spontaneous activation of NMDAR increased recording
noise and produced a small tonic depolarization which was similar in both
areas. We conclude that this basic property of excitatory transmission is
conserved across PFC and VC synapses and is therefore unlikely to contribute
to differences in firing patterns observed in vivo in the two regions. |
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INTRODUCTION |
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-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptors at excitatory synapses. Because these receptors have distinct
kinetics (AMPA, 
= 2–7 ms; NMDA, = 50–100 ms) and
because the NMDA receptor is important for
Ca2+-dependent plasticity and learning
(Bliss and Collingridge 1993
;
Malenka and Nicoll 1999), the
ratio of NMDA receptor (NMDAR) mediated to AMPA receptor (AMPAR) mediated
currents at excitatory synapses is likely to be a key determinant of both the
short-term integration of synaptic inputs by cortical neurons and their
long-term modification. It has been shown that in visual cortical synapses in
vitro, neural activity coregulates the AMPA and NMDA components of excitatory
postsynaptic currents (EPSCs) to maintain a constant ratio
(Watt et al. 2000). However,
little is known as to whether this coupling of NMDARs and AMPARs is regulated
in the same way across cortical areas. Although cortical areas can differ in
terms of architectonics and cell types, it is not known whether there are
regional differences in basic excitatory transmission. Synapses in different
cortical areas may be subject to different activity regimes, and synaptic
transmission could be tailored to accommodate this, providing a substrate for
different processing demands of local circuitry.
Regional variations in the NMDA/AMPA ratio have been proposed to contribute
(Compte et al. 2000;
Lisman et al. 1998; Wang
1999,
2001) to the ability of
prefrontal cortical neurons to exhibit persistent activity, a candidate neural
correlate of short-term memory in primates
(Chafee and Goldman-Rakic 1998;
Fuster and Alexander 1971;
Miller et al. 1996; Romo et al.
1999) and rodents (Sakurai and
Sugimoto 1986; Schoenbaum and Eichenbaum
1995a,b;
Jung et al. 1998;
Kesner et al. 1996;
Ragozzino and Kesner 2001).
The NMDAR is a favored candidate mechanism for sustained recurrent excitation
because the slow kinetics aid stability of the memory trace, and the dual
activation requirements of depolarization and ligand binding could be used to
select a reverberating circuit of only those cells which receive an initial
stimulus. Support for such models has so far been indirect. Higher levels of
mRNA for NMDARs in prefrontal cortex than in other cortices were found in a
human postmortem immunohistological study
(Scherzer et al. 1998), and in
rats, antagonists for NMDARs, given either systemically
(Verma and Moghaddam 1996) or
through microinjection of the PFC (Aura and
Riekkinen 1999), impair performance on delayed alternation tasks
requiring working memory.
Here we attempt to assess a possible specialization of excitatory
transmission in the PFC by comparing it to transmission in a functionally and
anatomically distinct cortical region using precisely the same methods. The
primary visual cortex is a well-studied area in rat, not thought to be
involved in working memory processes. Prior studies have measured the
NMDA/AMPA ratio in both visual cortical culture and slices
(Umemiya et al. 1999;
Watt et al. 2000). In
comparison, although specializations in synaptic plasticity have been found in
the prefrontal cortex (PFC) (Hempel et al.
2000), less is known about the functional NMDA/AMPA ratio. Burgard
and Hablitz (1993) assessed the
contribution from NMDARs to excitatory synaptic transmission in PFC layer 2/3
pyramidal neurons during early postnatal development (p3–p14). Layer 2/3
is a good candidate for a layer in which reverberatory circuits of recurrent
excitation may be found, since it possesses strong lateral excitation
(Lewis and Gonzalez-Burgos
2000). Surveying layer 2/3 synapses over a longer developmental
range is warranted, since the function of NMDAR at cortical synapses is
developmentally regulated, in part by a change in receptor subunit expression
which occurs around p14 in visual and somatosensory cortex
(Flint et al. 1997;
Nase et al. 1999;
Sheng et al. 1994). By around
p28, many other properties of cortical synapses have largely matured
(Reyes and Sakmann 1999).
Therefore to examine the issue of NMDAR and AMPAR regulation across cortical areas, and to explore the hypothesis that NMDARs make a greater contribution to transmission in the PFC, we measured the NMDA/AMPA ratio in layer 2/3 pyramidal neurons in slices from 2- to 4-week-old rats using several complementary approaches. We found that the relative contributions of NMDARs and AMPARs were remarkably conserved between the two cortical regions. The similarity across cortical regions was true for mixed NMDAR- and AMPAR-mediated miniature EPSCS (mEPSCs), for evoked EPSCs, and for spontaneous activation of synaptic and extrasynaptic NMDARs in the absence of AMPAR-mediated transmission. These results indicate that the function of AMPARs and NMDARs are regulated similarly in the supragranular layers of two functionally distinct cortical regions, suggesting a homogeneity of regulation of these receptors across the cortex. Therefore specializations of these regions for particular patterns of activity (such as persistent activity) may more likely reflect regional differences in input, modulation, or circuitry.
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METHODS |
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), using the shape of the
subcortical white matter as the primary landmark. The anterior/posterior
position of PFC slices corresponded to plates 9–11 of the atlas; VC
slices corresponded to plates 41–48. About six slices from each
hemisphere equilibrated undisturbed at room temperature for at least 1 h,
bubbled with 95% O2-5% CO2, before recording. Voltage-clamp recording
Glass micropipettes were pulled from 1.0 mm outer diameter thin-walled
capillary tubing (Warner Instruments) on a Flaming-Brown horizontal puller
(Sutter). For mEPSC experiments, internal solution contained the following (in
mM): 130 K-methylsulfonate or K-gluconate, 10 KCl, 10 HEPES, 0.5 EGTA,
2–3 Na2ATP, 0–1 guanosine 5'-triphosphate (GTP),
and 2 MgSO4. Osmolarity was 280–290 m
and pH
7.2–7.4. For evoked EPSC experiments, internal solution contained the
following (in mM): 130 cesium methanesulfonate, 10 KCl, 10 HEPES, 1
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic
acid (BAPTA), 2–3 Na2ATP, 0–1 GTP, 2 MgSO4,
10 tetraethylammonium (TEA)-Cl. Whole cell recording pipette resistances were
2.5–5 M. Pyramidal cells of layers 2/3 were illuminated and
visualized using a x40 water-immersion objective mounted on a
fixed-stage phase contrast microscope (Nikon Optiphot UD), and the image was
displayed on a video monitor using a charge-coupled device camera.
Voltage-clamp recordings were performed using an Axopatch 1D amplifier (Axon
Instruments). Signals were filtered at 2 kHz. Recordings were made 
8 h
following slicing.
Acquisition
Signals were sampled at 5–10 kHz and analyzed using the IgorPro (Wavemetrics, Oswego, OR) data acquisition software and customized analysis procedures running on a Macintosh G3 or G4 computer.
mEPSCS. Recordings were made in ACSF containing no added
Mg2+ to reduce Mg2+ block of
NMDARs. Tetrodotoxin (1 mM) and bicuculline (50 µM) were added to isolate
excitatory mEPSCs. Glycine (50 µM), a necessary cofactor for NMDAR
activation, was also added. Slices were transferred to the recording chamber
from 2 mM Mg2+ ACSF and equilibrated for 
25 min
before recordings were attempted, to allow the Mg2+ to
wash from the slice. Under these conditions, mEPSCs having both an AMPAR- and
an NMDAR-mediated component were readily observed. Ten to twenty 30-s traces
were acquired for each neuron. All recordings were done at room temperature to
lower the frequency of events and therefore decrease the likelihood that
successive events would occur within the long NMDAR-mediated tails of the
mEPSCs. In some cases a second round of acquisition proceeded after bath
perfusion of 100 µM APV, to isolate AMPA currents.
EVOKED EPSCS. Mixed NMDA/AMPA EPSCs were evoked in 2 mM
Mg2+ ACSF with bicuculline (50 µM) and glycine (50
µM). The slice was stimulated with a 100-µs shock delivered through a
glass micropipette (tip diameter 10–50 µm) and placed 50–100
µm lateral to the recording pipette in layer 2/3. Stimulus amplitude was
adjusted to produce a single-peaked response with a short constant latency
(2–3 ms) and an average amplitude of approximately 60 pA. Visually
identified pyramidal cells were held at –70 mV. Recordings began 10
min after initiating whole cell recording, to allow dialysis of the
Cs+/TEA internal solution, so that large depolarizing voltage steps
could be given while minimizing activation of voltage-dependent K+
currents. Equilibrium of dialysis was seen on average as a stable
approximately twofold increase in resting input resistance. Evoked EPSCs were
recorded at –90, 0, and +50 mV, and in some cells intervening voltage
steps. EPSCs were evoked at low frequency (once every 10–60 s) to
minimize accumulation of slowly decaying currents activated during steps to
+50 mV. We confirmed that this procedure did not result in long-term
potentiation (LTP) of the evoked EPSCs (perhaps because of the initial waiting
period of 10–12 min and the inclusion of 1 mM BAPTA in the recording
pipette) by comparing the EPSC amplitudes from the beginning and end of the
recording period.
NMDA "NOISE." In general, the slow time course and small amplitude of NMDAR-mediated mEPSCs makes it difficult to resolve individual events without the sharp rise time of an accompanying AMPA mEPSC as a "tag." Yet detection using only mEPSCs which also have an AMPA component excludes any all-NMDAR-mediated events ("silent synapses"), as well as contributions from extrasynaptic NMDARs which could also differ in the two regions. To monitor the contribution of NMDARs not accompanied by AMPAR-mediated transmission, we compared epochs of data recorded in no added Mg2+ ACSF containing 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (20 µM), bicuculline (50 µM), glycine (50 µM), and TTX (1 mM), before and after blocking NMDARs with APV (100 µM).
Analysis
Cells were included if Vrest was more hyperpolarized
than –60 mV, and Rseries 20 M. Resting
(–70 mV) Rinput was monitored throughout the
recording; traces in which the resistance changed by >20% were excluded.
For recordings in normal internal solution, Rinput ranged
from 150 to 400 M; this was larger with Cs+ internal, due to
a blockade of K+ conductance, with a range of 300–800
M. Cs+ recordings also produced a depolarized
Vrest; in these cases Vrest was not
used as an inclusion criterion.
mEPSCs. Spontaneous events were measured using an in-house mEPSC analysis package which allows for consistent and automated analysis based on preset detection/selection parameters. The decays of average mEPSCs were fit with double-exponential functions. The peak of an mEPSC (taken as the AMPA peak) was measured as the mean current over a 1-ms window. NMDA current amplitude was measured over a 5-ms window from 18 to 23 ms after the AMPA peak, a time at which AMPA-only mEPSCs (n = 5) had declined to 0.26 ± 0.16 pA. The NMDA/AMPA ratio for mEPSCs was given as the ratio of these two measurements.
EVOKED EPSCS. Ten to twenty traces were averaged at each holding potential. Only single peaked, fast latency (2–3 ms) events were included to ensure monosynaptic responses. Occasional traces containing stimulus-evoked epileptiform events (reflecting inhibitory blockade with 20 µM bicuculline) were excluded from the average waveform. Average waveforms from the –90 and +50 mV holding steps were baseline subtracted and measured using in-house procedures written for IgorPro. The time of the peak current at –90 mV, considered to be fully mediated by AMPAR, was used to establish the time window for measuring the AMPA peak at +50 mV. The decay to baseline of the AMPA current at –90 was used to select a time window for measurement of the NMDA current; a 10-ms measurement window beginning 40 ms after the stimulus artifact was used. This current was designated as the NMDA measurement. (INMDA at +50mV/IAMPA at +50mV) was taken as the NMDA/AMPA ratio.
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RESULTS |
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For rats ages p16–p21, mixed NMDAR- and AMPAR-mediated mEPSCs were well resolved in nominally Mg2+-free ACSF (Fig. 1A). mEPSC amplitudes varied from our threshold of 7 pA up to 100 pA. The average number of mEPSCs collected per cell was 190 ± 16. Averaging events produced mEPSC current traces well fit with double exponential functions. The slow component of the mEPSCs was abolished with bath application of 200 µM APV (Fig. 1B).
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The average mEPSC amplitude did not differ significantly between brain areas (PFC: 19.1 pA ±1; VC: 17.5 ± 0.7). No statistically significant difference was found in the average NMDA/AMPA ratio between the PFC and VC. Indeed, the grand average mEPSC waveforms from each area (an average of averages from each cell) were quite similar (Fig. 2A). The NMDA/AMPA ratio for the PFC was 27% (±1%, n = 27); for the VC, 28% (±3%, n = 28).
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Although the proportion of peak current from NMDARs was indistinguishable
in both brain areas, it is possible that a regional difference in NMDAR
kinetics—similar to that caused by the developmental change in the
relative abundance of NR2B and NR2A (Flint
et al. 1997; Nase et al.
1999; Sheng et al.
1994)— could give rise to different functionality in the two
areas. However, double exponentials fit to the average mEPSCs from each cell
showed no appreciable difference in decay kinetics between the two areas
(Fig. 2Ab).
Although the NMDA/AMPA ratios between areas were indistinguishable, it
could be the case that each area has a heterogeneous population of excitatory
connections, with varying NMDA/AMPA ratios found across synapses. It is
possible that the PFC may have a specialized subset of high-NMDAR synapses
which subserve persistent activity, but such regional differences in the
proportion of high and low NMDAR synapses may be lost in averaging. Therefore
40 mEPSCs were randomly selected from each cell, and the AMPA peak of each
event was plotted against its NMDA measurement. A fit to the data showed a
similar degree of correlation of AMPA-to-NMDA responses in the two areas (PFC,
r = 0.48; VC, r = 0.57;
Fig. 2B). As with the
average NMDA/AMPA ratios, the distributions of the NMDA/AMPA ratios of
individual events did not significantly differ (Kolmogorov-Smirnov test,
P = 0.65). The degree of correlation observed is qualitatively
similar to findings from Watt et al.
(2000). The slightly higher
mean correlation of 0.65 reported in that study may reflect the lower number
of cells pooled (n = 6), a difference between cell types (layer IV in
the prior study vs. layer 2/3 in the present study), or a greater degree of
variation due to the comparatively smaller NMDA signal in our
measurements.
Although the results of the mEPSC analysis showed no difference for ages p16–20, we duplicated the mEPSC experiments using rats from p26–29. Very similar results as those from Fig. 2, A and B, are shown in Fig. 2, C and D, for the older animals. Again, there were no statistical differences between the two areas, with both having an NMDA/AMPA ratio of approximately 30% and decay kinetics of approximately 65 ms, essentially the same as with the younger group. Also, the correlations of NMDA versus AMPA measurements were similar to those observed in the p16–20 rats.
Evoked EPSCs
Analysis of mEPSCs provides the advantage of relatively unbiased sampling from a large number of synapses onto a single cell. However, due to the noisiness of the baseline in 0 Mg2+ recordings, the signal-to-noise ratio is low in comparison with AMPA-only mEPSCs, raising the possibility that small but real differences could be missed. Therefore we corroborated our results by using extracellular stimulation to evoke larger and stimulus-locked EPSCs. Another important advantage is that evoked EPSCs may include currents mediated by NMDA-only synapses.
In Mg2+-free ACSF with bicuculline but no TTX, slices tended to become active and fire frequent epileptic discharges which interfered with clean recordings. Therefore we adopted a different strategy for the evoked EPSCs. Slices were perfused in normal (2 mM) Mg2+ so that at hyperpolarized potentials most NMDA current was blocked. We assessed the time course of the AMPA component by stimulating while the cell was held at –90 mV. The slice was then stimulated while the cell was held to +50 mV, which provides sufficient depolarization and driving force to reveal the NMDAR component of the EPSC (Fig. 3A). EPSCs reversed at or slightly above 0 mV and were completely blocked by the addition of CNQX and APV (not shown). Averages of 10–20 responses at both –90 and +50 mV were used to measure the AMPA and NMDA components. As with the mEPSCs, there was no difference in the NMDA/AMPA ratio between the two areas (PFC: 125% ±6, n = 9; VC: 125% ±7, n = 10). This method somewhat overestimates the AMPA component, since there was also a small degree of activation of NMDA at the time of the AMPA peak. Measurements from isolated NMDA currents indicate that this accounted for on average 22% of the measured peak (±8%, n = 4) and therefore we estimate the NMDA/AMPA ratio at +50 in the evoked condition to be approximately 160%.
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Factors contributing to discrepant estimates of the NMDA/AMPA ratio
The NMDA/AMPA ratio measured from evoked EPSCs (160%) was much larger than
that measured from mEPSCs (30%). Several methodological differences could
account for this discrepancy. First, since we are only evaluating mEPSCs which
have an AMPA component, we could be neglecting a large population of NMDA-only
(silent) synapses. However, silent synapses in visual cortex are reported to
be largely absent by the ages studied
(Rumpel et al. 1998).
Second, although we used a nominally Mg2+-free ACSF
for the mEPSC recordings, endogenous Mg2+ from the
slice, or Mg2+ from the internal solution (from either
inside the cell or blown onto the cell during patching), could provide a
partial block of NMDARs (Hestrin et al.
1990) sufficient to greatly decrease the NMDA/AMPA ratio. To test
this, we recorded evoked NMDA-only EPSCs in a range of
Mg2+ concentrations. Although disinhibited by
bicuculline, the inclusion of CNQX decreased the likelihood of epileptiform
activity (although some epileptiform discharges, presumably mediated solely
through NMDARs, did occasionally occur). NMDA-only events were recorded over a
range of voltages (–90, –70, –50, –30, 0, +30, +50,
+70 mV), and I/V curves generated for each of four
Mg2+ concentrations: 2, 1, 0.2 mM, and no added
Mg2+ (example, Fig.
4A). In all cases, the I/V curve had the classic
"j-shape" characteristic of the Mg2+-based
voltage dependence (Fig.
4B). To quantify the degree of Mg2+
block in the no added Mg2+ condition, I/V
relations for the known values of [Mg2+] were then fit
with the equation
 | (1) |
). The
best fit of this equation to the "0" Mg2+
curve gave an estimate of the actual residual Mg2+, 0.03
(±0.01) mM. To test the validity of this estimate, we recorded mEPSCs
in 0.03 mM Mg2+. The average waveforms of the two
Mg2+ conditions (no added Mg2+ and
0.03 mM Mg2+) were indistinguishable
(Fig. 4C).
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To estimate the reduction of the NMDA component in mEPSCs due to residual Mg2+, we fitted a line to all data points for all voltage steps more positive than 0 mV (dashed line, Fig. 4B) and measured the difference in current at –70 mV on this line and at the corresponding point on the "0" Mg2+ curve. This comparison showed that the 0 Mg2+ curve evaluated at –70 mV was reduced to 62% of what it would have been had the NMDA I/V relation been linear (i.e., no Mg2+ block). Taking this into account, the corrected estimate should put the NMDA/AMPA ratio for the mEPSCs at 45% for both the PFC and VC.
Just as the NMDA/AMPA ratio may have been underestimated in the mEPSCs due
to residual Mg2+, it is possible we may have
overestimated the NMDA/AMPA ratio of the evoked EPSCs. One way this could
happen is if there was greater dendritic filtering in the evoked EPSCs, which
would tend to diminish the faster AMPA events more than the slow NMDA events.
We do not think this is likely, since the average EPSC rise times at –90
mV and the NMDA/AMPA ratios were poorly correlated (r = –0.31),
and the NMDA/AMPA ratios from cells with the fastest average AMPA rises
(<1.5 ms, n = 9; average ratio = 127 ± 7%) were not on
average different from the ratios of the remaining cells (>1.5 ms,
n = 10; average ratio = 123 ± 7%; P = 0.3),
suggesting that filtering does not account for the large NMDA/AMPA ratio in
the evoked EPSCs. Another possible cause for overestimating the ratio is if
the AMPA component were inwardly rectifying at depolarized potentials
(Kumar and Huguenard 2001). To
test this, we produced an AMPA-only I/V curve (in APV and
bicuculline) over the same range of voltages given above. Beginning around +30
mV there was on average some inward rectification (example,
Fig. 4D). We measured
the inward rectification at +50 mV (the voltage at which the NMDA/AMPA ratio
had been assessed in the EPSCs) and found the response diminished to 68%,
based on a linear fit of the AMPA conductance at hyperpolarized potentials
(dashed line, Fig.
4D). Since the AMPA component was diminished to 68% of
maximum, this corrects the NMDA/AMPA ratio for the evoked EPSCs from 160 to
109%.
These two methodological corrections—residual Mg2+ and inward rectification of AMPA at depolarized potentials—lessens the discrepancy between the NMDA/AMPA ratio as measured with mEPSCs versus evoked EPSCs, but still leaves a difference of approximately 45% versus approximately 109%. A number of other factors may account for the remaining discrepancy and are explored in DISCUSSION.
NMDA "noise"
Although the mEPSCs and EPSCs suggest an equal NMDA/AMPA ratio in PFC and
VC, these techniques do not assess possible activation of extrasynaptic NMDA
receptors by ambient glutamate or glutamate spillover
(Clark et al. 1997;
Rusakov and Kullmann 1998).
Previous studies have shown that in 0 Mg2+ NMDARs
contribute a tonic, inward, noisy current in cortical pyramidal neurons
(LoTurco et al. 1990). We
compared this small "NMDA noise" in the PFC and VC as a means to
capture all NMDA conductances: at mixed NMDA and AMPA synapses, silent
synapses, and extrasynaptic NMDARs. If there is increased extrasynaptic NMDAR
activation in the PFC, this assessment should capture it. We found that bath
application of the highly specific NMDAR blocker APV (100 µM) reduced noise
and abolished a tonic inward current (Fig.
5A). However, the change in the average holding current
after APV was similar in both areas (PFC: –5.9 ± 1.0 pA,
n = 7; VC: –4.8 ± 1.5 pA, n = 5) and not
statistically different (Fig.
5B). The noise, measured as the SD of the fluctuating
inward current at –70 mV, was also decreased to a similar degree in both
brain areas. This suggests that extrasynaptic NMDARs and silent synapses are
not differentially distributed between PFC and VC layer 2/3 at this age.
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DISCUSSION |
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The NMDA/AMPA ratio was markedly different depending on whether mEPSCs or
evoked EPSCs were assessed. This discrepancy might be accounted for in several
ways. First, due to our method of mEPSC detection, only those mEPSCs which
have an AMPA component ("AMPA tagged") are selected, because slow
rise times make NMDA-only events difficult to detect. Therefore we are
excluding any silent synapses, which could account for more NMDA current
measured with evoked EPSCs. Silent synapses would work particularly well in
NMDA-based models of persistent activity, but we think it unlikely that this
accounts for the discrepancy. Studies characterizing the development of silent
synapses in the visual cortex (Rumpel et
al. 1998) find a general elimination of silent synapses in VC by
p14. Nevertheless, small-AMPA/big-NMDA mEPSCs may also be missed and cause an
underestimate of the NMDA contribution. Another difference between the mEPSCs
and evoked EPSC recordings is that the latter were recorded with internal
solutions containing high cesium, which may diminish sensitivity of NMDARs to
Mg2+ blockade (Qian
et al. 2002). However, this is unlikely to account for the
observed difference, since Mg2+ blockade is largely
absent at the depolarized voltage (+50 mV) used.
More probable factors giving rise to the discrepancy are those which
directly reflect properties of spontaneous vesicle fusion at individual
synapses versus release evoked (nearly) synchronously at many synapses.
Recruitment of a large subset of synapses onto a postsynaptic cell during
extracellular stimulation may result in glutamate spillover which can
contribute to the NMDA component in the EPSCs
(Clark et al. 1997;
Rusakov and Kullmann 1998).
Also, variations in onset time of component EPSCs (due to variations in
synaptic delays, axonal path lengths, and fiber recruitment thresholds) can
smear out the time course of compound EPSCs relative to that of single release
site events. Such smearing out of the response may favor summation of the far
slower NMDA component, thereby artificially inflating the NMDA/AMPA ratio.
Consistent with this possibility, evoked EPSCs had much longer durations and
slower rise times than mEPSCs.
For these two reasons we suspect that the NMDA/AMPA ratios measured with mEPSCs or EPSCs are likely to be different in the way we have observed. Indeed, a review of the related literature seems to indicate this. For the purposes of close comparison with other studies, we have generated a partial survey of reported NMDA/AMPA ratios and our ratios in Table 1, along with other relevant parameters. In the final column of the table we have attempted to normalize the various findings by using the reported parameters and Eq. 1 to estimate the ratio of maximum conductance of NMDA to AMPA (given as gmax ratio). Interestingly, as in our study, ratios assessed with mEPSCs tend to be smaller than those assessed in pairs or evoked EPSCs, with two qualifications. First, in evoked current-clamp recordings (first 2 rows) gmax ratio values are notably higher. This may reflect the fact that the somatic and subsynaptic membrane may be further from isopotential during current-clamp recordings. Second, in the mEPSC data, there may be an age dependence to the gmax ratio, in that the first two rows show a larger gmax ratio but are sampled in younger animals (p1–p15). Generally, the range of NMDA/AMPA ratios reported is consistent with our findings.
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A number of factors are relevant to the applicability of these results to
NMDA-based models of persistent activity. First, it is not clear that the same
mechanisms or structures are implemented in primate and rodent PFC; for
example, layer 4 is found in the PFC of primates but not in rodents. Yet there
is evidence for the prefrontal cortex as important for working memory in rat
as with monkey (e.g., that PFC lesions in rat impair performance on delayed
alternation tasks requiring working memory)
(Sakurai and Sugimoto 1985).
Although persistent activity and delay-tuned cells have best been explored in
the PFC of primates, growing evidence in rat PFC links delay period firing and
items held on-line in memory (Jung et al.
1998; Kesner et al.
1996; Ragozzino and Kesner
2001; Sakurai and Sugimoto
1986; Schoenbaum and Eichenbaum
1995a,b).
We confined our recordings to layer 2/3 pyramidal cells. Superficial layers
of the cortex have rich local recurrent excitation
(Lewis and Gonzalez-Burgos
2000) and as such may be good candidates for local reverberatory
working memory circuits. However, it is possible that excitatory synapses of
layer 2/3 are not those involved in recurrent excitation leading to persistent
activity. Also, NMDAR- and AMPAR-mediated currents have been shown to be
independently regulated by a variety of neuromodulatory agents
(Aramakis and Metherate 1998;
Arvanov et al. 1997;
Arvanov and Wang 1998;
Castro et al. 1999;
Seamans et al. 2001). Future
experiments could investigate the effect of such substances on the NMDA/AMPA
ratio as measured in mixed NMDA/AMPA mEPSCs or EPSCs in PFC and elsewhere.
The similarity of layer 2/3 NMDA contribution in PFC and VC does not
support claims that associative areas specialized for persistent activity
should have a greater contribution from NMDARs than primary sensory areas.
However, the large NMDA/AMPA ratio (approximately 109%) in the evoked EPSCs
suggests there is ample NMDA conductance in both areas to provide stability of
the memory trace in the models. Therefore NMDARs may be abundant throughout
the cortex and persistent activity may indeed be maintained by recurrent
excitation via NMDAR-mediated current; however, whether such activity is seen
may be strongly dependent on the local architecture. Miller and Cohen
(2001) have characterized
these two accounts of persistent activity as "cellular" versus
"circuit-based." Although the PFC has been generally targeted as a
key region for working memory, recent results showing delay period activity in
primary visual cortex of monkey during a visual task
(Super et al. 2001) raise the
possibility that comparable and large NMDA contributions in the VC as well as
PFC may contribute to persistent activity in both regions.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests: S. Nelson, Brandeis University, MS 008, 415 South St., Waltham, MA 02454-9110 (E-mail: nelson{at}brandeis.edu).
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