 |
INTRODUCTION |
Layer I of the cerebral cortex is a thin sheet of tissue containing dense neuropil but relatively few neurons. It is often called the molecular or cell-free layer (DeFelipe and Jones 1988
; Peters and Yilmaz 1993
). Immunohistochemical studies have demonstrated that most layer I neurons are
-aminobutyric acid positive, suggesting they are inhibitory neurons (Gabbott and Somogyi 1986
; Li and Schwark 1994
). We recently demonstrated that layer I neurons have repetitive firing properties typical of interneurons (Zhou and Hablitz 1996a
,b
). In rat visual cortex, layer I neurons make and receive a large number of synapses, suggesting that they are actively involved in cortical information processing (Beaulieu and Colonnier 1985
; Beaulieu et al. 1994
). However, because of technical difficulties, pharmacological and biophysical studies of synaptic inputs to layer I neurons are lacking.
Morphological studies of layer I neurons in which intracellular biocytin staining was used have shown these neurons to have extensive axonal arbors (Zhou and Hablitz 1995
). Excitatory inputs from underlying pyramidal cells or other areas would be expected to impinge on layer I inhibitory neurons. Layer I cells may in turn inhibit pyramidal neurons via synapses onto their dendrites and/or somata. Therefore layer I neurons can serve as feedback or feedforward inhibitory elements. Because of the extensive axonal arborization, excitation of a single layer I neuron may result in inhibitory influences on neurons in a relatively large area. Layer I neurons may also interact with each other and/or with deeper layer inhibitory neurons, resulting in disinhibition.
Inhibitory interneurons in deeper cortical layers (Geiger et al. 1995
; Jonas et al. 1994
) and in the hippocampus (Koh et al. 1995
) have been shown to express
-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptors with rapid kinetics and high Ca2+ permeability. The kinetic properties of AMPA-receptor-mediated synaptic currents on inhibitory interneurons have been suggested to differ from those of principal cells (Hestrin 1993
). Therefore as an important step toward understanding neocortical layer I, we have characterized the biophysical and pharmacological properties of spontaneous excitatory postsynaptic currents (sEPSCs) in rat neocortical layer I neurons. With the use of the whole cell patch recording technique combined with visualization of neurons in brain slices, we found that sEPSCs in layer I neurons are principally mediated by AMPA receptors. Under optimal recording conditions, these excitatory postsynaptic currents (EPSCs) have rapid kinetics and are inwardly rectifying. Preliminary results have appeared in an abstract (Hablitz and Zhou 1995
).
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METHODS |
Slice preparation
Sprague-Dawley rats (14-20 days old) were used in this study. All animals were housed and handled according to approved guidelines. Thin brain slices were prepared according to methods described previously (Edwards et al. 1989
; Zhou and Hablitz 1996a
). Briefly, after decapitation the brain was rapidly dissected out and immediately immersed in ice-cold saline. Brain slices (200-µm thick) were then cut from frontal cortex on a Vibratome. In some experiments slices were also obtained from cingulate cortex (Paxinos and Watson 1986
). Slices were placed in a storage chamber at room temperature (22 ± 1°C, mean ± SD) for 1 h before recording and were used up to 8 h after preparation. The normal extracellular bathing solution contained (in mM) 125 NaCl, 3.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 26 NaHCO3, and 10 D-glucose. The bathing solution was continuously bubbled with 95% O2-5% CO2 to maintain pH ~ 7.4. Slices were perfused at a rate of 4 ml/min, resulting in a complete solution exchange within 5 min.
Bicuculline methiodide (Sigma), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, Tocris Neuramin), and D(
)2-amino-5-phosphonovaleric acid (D-APV, Tocris Neuramin) were prepared as 10 mM stock solutions. They were then frozen and added in small aliquots to the physiological bathing solution during the experiment. Final concentrations of CNQX, D-APV, and bicuculline methiodide were 10, 20, and 10 µM, respectively. Tetrodotoxin (TTX) and glycine were from Sigma. Cyclothiazide (CTZ) was a gift from Eli Lilly. All recordings were made in the presence of 10 µM glycine and 10 µM bicuculline methiodide.
Whole cell recording
With the use of Nomarski optics and a ×40 water-immersion lens, layer I neurons were reliably identified during recording by their location below the pial surface (Zhou and Hablitz 1996a
). All recordings were made at room temperature (22 ± 1°C). Patch electrodes were prepared from Garner KG-33 glass with the use of a Narishige PP-83 puller. Electrodes were coated with Sylgard. Series resistance (Rs, defined here as the total resistance between the amplifier and the cell interior) was estimated according toRs = 10 mV/I, where I was the current (filtered at
10 kHz) evoked by a 10-mV pulse when the pipette capacitance was fully compensated. During actual recordings, Rs was between 3 and 12 M
and was not compensated. Rs was continuously monitored and recordings were terminated when Rs was
12 M
or a significant increase occurred. The intracellular solution contained (in mM) 10 KCl, 125 CsCl, 2 N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314), 10 N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid, 2 Mg-ATP, 0.2 sodium guanosine5
-triphosphate, and 0.5 ethylene glycol-bis(
-aminoethyl ether)-N,N,N
,N
-tetraacetic acid, pH adjusted to 7.3 with KOH. A KCl-based intracellular solution was also used in some cells and QX-314 was omitted in about one third of the recordings. Osmolarity was adjusted to 270-290 mosM. Tight seals (
3 G
before break into whole cell mode) were achieved by applying positive pressure to the pipette during the approach to the cell. Before tight seals were formed, all offsets were nulled. The liquid junction potential after going whole cell was not corrected.
Electrical signals were recorded with the use of an Axopatch-200 amplifier (Axon Instruments) and stored on videotape. Signals were filtered (Frequency Devices) at 1-2.5 kHz and digitized at 0.05 ms per point for capturing individual events and at 0.3 ms per point for obtaining long stretches of recordings. Digitization and analysis of the records was achieved with the use of SCAN software (courtesy of J. Dempster, University of Strathclyde, Glasgow, Scotland). Automatic measurements of the rise time and decay time constants of synaptic currents were manually verified and corrections were made when necessary.
Data base and analysis
In the following analysis, 80 layer I neurons (67 from frontal cortex and 13 from cingulate cortex) were examined. No difference in sEPSCs was noticed between the two groups of layer I neurons and the data were pooled. No difference was observed between recordings obtained with and without intracellular QX-314 and these results were pooled. In individual cells, the number of spontaneous synaptic events recorded varied from 10 to >5,000 depending on frequency and recording time. The decay of synaptic currents was fitted to the following function: I(t) = Af exp(
t/
f) + As exp(
t/
s) + C, where I(t) was the amplitude of EPSCs at time t, Af and As were the amplitudes of the fast and slow components, respectively,
f and
s were the decay time constants of the fast and slow components, respectively, and C was the residual current at the end of the fitting interval. Results are expressed as means ± SD. Correlation coefficients (r) were calculated with the use of a least-squares linear regression analysis.P
0.01 was considered statistically significant.
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RESULTS |
AMPA-receptor-mediated sEPSCs
sEPSCs were recorded in the absence of TTX with the use of holding potentials of
60 or
70 mV, values close to the resting membrane potential (RMP) of neocortical layer I neurons (Zhou and Hablitz 1996a
,b
). sEPSCs recorded in the presence of 0.5 µM TTX were considered miniature EPSCs (mEPSCs, i.e., quantal synaptic events). The frequency of sEPSCs and mEPSCs at RMP varied considerably among the cells, possibly because of differences in the preservation of synaptic connections in different cells. Frequency histograms of interevent intervals were approximated by single-exponential functions, indicating that recorded sEPSCs and mEPSCs were random events. sEPSC frequency was 1.3 ± 0.35 Hz (n = 26, range 0.2-5 Hz), with mEPSCs being slightly less frequent.
At RMP, in a bathing solution containing 1.3 mM Mg2+, bath application of 20 µM D-APV had no effect on the waveform or amplitude of mEPSCs (Fig. 1A). Spontaneous synaptic activity was completely blocked by bath application of 5-10 µM CNQX. These results indicate that, at RMP and in the presence of physiological extracellular Mg2+, sEPSCs and mEPSCs are primarily mediated by AMPA receptors.

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| FIG. 1.
Basic properties of spontaneous excitatory postsynaptic currents (sEPSCs) in neocortical layer I neurons. A: at 70 mV, sEPSCs and miniature excitatory postsynaptic currents (mEPSCs) are mediated predominantly by -amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptors and contribution from N-methyl-D-aspartate (NMDA) receptors is minimal. A1: averaged mEPSC at 70 mV recorded in the presence of 0.5 µM tetrodotoxin (TTX) and 1.3 mM Mg2+. A2: bath application of 20 µM D( )2-amino-5-phosphonovaleric acid (D-APV) did not significantly alter averaged mEPSC waveforms. B1: NMDA component in averaged mEPSCs was minimal at 60 mV in normal bath solution. B2: depolarization to 30 mV revealed a slow NMDA component. C: averaged mEPSC at 70 mV in Mg2+-free bath solution. Note this mEPSC had both fast and slow components. C2: block of the fast component by 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). D: averaged mEPSC with dual components recorded in Mg2+-free bath solution. D2: slow component was blocked by 20 µM D-APV. Records in A-D were taken from 4 different layer I cells. Traces are averages of 17-160 individual mEPSCs. Decays were fit with double-exponential functions. Fits are shown superimposed and time constants are given below each trace. f, decay time constant of the fast component; s, decay time constant of the slow component.
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N-methyl-D-aspartate (NMDA) EPSCs were unmasked by depolarization or Mg2+-free solutions. Figure 1B1 shows an averaged mEPSC recorded in the presence of 1.3 mM extracellular Mg2+. A small slow component was observed at a holding potential of
60 mV. When the holding potential was changed to
40 mV or more positive, the averaged mEPSC displayed a larger slow component (Fig. 1B2). Similar results were obtained in all layer I neurons tested(n = 11). Bath application of 20 µM D-APV eliminated the slow component seen at depolarized levels (n = 5), indicating that it was mediated by NMDA receptors. In nominally Mg2+-free solution, mEPSCs were composed of fast and slow components (Fig. 1, C and D). In eight layer I neurons tested, the fast component of averaged mEPSCs was blocked by 10 µM CNQX (Fig. 1, C1 and C2), whereas 20 µM D-APV blocked the slow component (Fig. 1, D1 and D2). Results similar to those described above were also obtained with sEPSCs (n = 4). These results clearly indicate that layer I neurons have dual-component EPSCs.
Rapid kinetics of AMPA-receptor-mediated mEPSCs in layer I neurons
When recording conditions were optimal (Rs < 7 M
) (n = 21), mEPSCs in layer I, recorded in the presence of 20 µM D-APV to eliminate contributions from NMDA receptors, had very rapid kinetics, as shown in Fig. 2. The averaged mEPSC had a 10-90% rise time (T10-90) of 0.25 ms (Fig. 2A). Most individual mEPSCs also had T10-90s of ~0.25 ms (Fig. 2, B and C). A double-exponential function with a fast and slow term was needed to fit the decay of the averaged mEPSC (Fig. 2A). The fast component had a decay time constant (
f) of 0.71 ms and comprised 85% of the total amplitude. The slow component had a decay time constant (
s) of 3.53 ms. For the majority of individual mEPSCs, the decay was also double exponential with a dominant fast and a small slow component (Fig. 2B). However, there was considerable variation in the decay parameters among individual mEPSCs; the contribution to the total amplitude from the slow component ranged from ~0 to 45%. Table 1 summarizes the kinetic properties of AMPA-receptor-mediated mEPSCs in 21 layer I neurons in which recording conditions were optimal.

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| FIG. 2.
mEPSC kinetics are rapid in layer I neurons. Recordings were made at a holding potential of 70 mV in the presence of 20 µM D-APV. Series resistance (Rs): 3 M . Signals were filtered at 2.5 kHz and sampled at 0.05 ms per point. A: averaged mEPSC from this cell. Events with 10-90% rise time (T10-90) >0.35 ms and/or decay time constants longer than twice the average values were not included. Double-exponential fits are shown superimposed and the kinetic parameters are listed below each trace. B: individual mEPSC from this cell. C: distribution of T10-90s in this cell (n = 210). Binwidth: 0.05 ms.
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Recording conditions had substantial effects on the waveforms of recorded mEPSCs. In a group of 32 layer I neurons where Rs was 7-12 M
, the kinetics of mEPSCs were significantly slower than those listed in Table 1. For averaged mEPSCs that included all events, T10-90 was 0.7 ± 0.06 ms (n = 32). The decay of averaged mEPSCs was also best described by double-exponential functions.
f was 2.45 ± 0.19 ms (89 ± 3% of the total amplitude);
s was 13 ± 2.2 ms. Similar observations were obtained for averaged sEPSCs (including all events) with a T10-90 of 0.72 ± 0.07,
f of 2.6 ± 0.22 (87 ± 5% of the total amplitude), and
s of13 ± 2.3 ms (n = 15).
Skewed mEPSC amplitude distribution
Amplitudes of mEPSCs varied considerably in all layer I neurons, ranging from 5 to 100 pA at RMP. In Fig. 3, mEPSCs were recorded at
60 mV and captured with a detection threshold near 3.5 pA. The amplitude histogram (Fig. 3A) suggests that amplitudes were not distributed in a single- or multimodal Gaussian fashion, as expected according to quantal theory (Katz 1966
). Instead, the amplitude distribution was skewed toward large events. Such skewed distributions were observed in all layer I neurons (n = 60) in which amplitude distribution histograms were constructed. Missed small and/or attenuated events could contribute to this skewness. However, 6- to 10-pA events were well above noise levels and still underrepresented in the amplitude histograms. Alternately, the broad amplitude distribution of mEPSCs might reflect intrinsic variations in quantal size. The lack of discrete peaks in the amplitude histograms could result from dendritic filtering.

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| FIG. 3.
Amplitude distribution ofmEPSCs and effects of dendritic cable filtering. A: amplitude distribution of mEPSCs.Events were recorded at 60 mV and captured with a detection threshold of 3.5 pA. Binwidth: 2 pA. Amplitude distribution was skewed to the right. B: plot of amplitude vs. rise time of mEPSCs. No significant correlation was observed. Note some small events had the fastest rise times, whereas some large events had slow rise times. These small fast events may arise from small quantal release at synapses close to the soma, whereas the large slow events may arise from large quantal release at distal synapses or from asynchronous transmitter release at proximal synapses. C: rise and decay time of mEPSCs are significantly correlated (r = 0.53), indicating a role of dendritic cable filtering. D: individual mEPSCs were selected and put intofast (T10-90 0.5, < 2 ms) and slow(T10-90 > 0.5 ms, > 2 ms) groups. Averaged fast mEPSC (trace 1) had a faster rise and decay time than averaged slow mEPSC (trace 2).
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Evidence for dendritic filtering of mEPSCs
Dendritic cable filtering effects on mEPSCs were first examined by plotting, for individual mEPSCs, rise time versus amplitude and decay time. Figure 3B shows that in this layer I neuron, T10-90s and mEPSC amplitudes were not correlated (r = 0.06; 1,993 events). This was true in all cells tested (r = 0.04 ± 0.03; n = 21). Note that in Fig. 3B some small-amplitude events had short rise times, whereas some large events had long rise times. Such a relation was seen in all cells in which similar plots were made(n = 21), suggesting that significant filtering was not present. However, unless all of the mEPSCs were generated at the soma or at synapses that had the same electrotonic distance to the soma, mEPSCs from different synapses must experience different dendritic filtering. Therefore it is more likely that other factors obscured the detection of dendritic cable filtering effects in the rise time-amplitude plots. For example, the small fast events could arise from small quantal release at synapses close to the soma, whereas the large slow events could arise from large quantal release at distal synapses or from asynchronous transmitter release at proximal synapses.
To further evaluate possible effects of dendritic filtering, decay time constants were plotted against rise times. For this analysis, the decay of individual mEPSCs was fitted to single-exponential functions for easier computation even though it left a small slow component unfitted. The time constants of such single-exponential fits were very close to the time constants of the fast components in double-exponential fits. Figure 3C shows that for mEPSCs the T10-90s were positively correlated with decay times (r = 0.53; 1,993 events). Similar results were obtained in all neurons tested (n = 21; r = 0.52 ± 0.11, range 0.31-0.72). To further demonstrate this relation, mEPSCs were sorted into fast (T10-90
0.5,
< 2 ms) and slow (T10-90 > 0.5 ms,
> 2 ms) groups. Averaged fast mEPSCs (Fig. 3D, trace 1) had a faster rise and decay time than averaged slow mEPSCs (Fig. 3D, trace 2). These results strongly suggest that the kinetics of somatically recorded mEPSCs were slowed by dendritic filtering. The modest r values indicate that the dendritic filtering effects may be nonlinear, as suggested by Spruston et al. (1993)
, and/or that other factors obscured dendritic cable filtering effects in plots such as those shown in Fig. 3C.
sEPSCs are more variable in amplitude and kinetics
The presence of mEPSCs with large amplitudes and slow kinetics suggested that dendritic cable filtering effects alone cannot account for all of the variations in mEPSCs. Because sEPSCs include both action-potential-dependent larger events and action-potential-independent smaller events, we reasoned that examination of properties of sEPSCs might provide insight into the origin of EPSC variability.
The amplitudes of sEPSCs, recorded in the absence of TTX, ranged from 5 to >200 pA, at holding potentials of
50 to
70 mV. Large-amplitude events presumably resulted from presynaptic action potentials triggering multivesicular release (Figs. 4C and 5A). Compared with those of mEPSCs, the kinetics of individual sEPSCs were more variable. Individual sEPSCs were assigned to three groups, as shown in Fig. 4, A and B. Group 1 included small, fast, presumably action-potential-independent events with T10-90s
0.7 ms (in this cell Rs was ~10 M
such that kinetics were relatively slow) and amplitudes
20 pA, group 2 contained slow, small events (T10-90 > 0.7 ms, amplitude
20 pA), and group 3 consisted of large, presumably action-potential-dependent events (amplitudes
60 pA). sEPSCs in each group were averaged and aligned by their rising phases as shown in Fig. 4A. The averaged sEPSCs were then scaled to the same peak size (Fig. 4B). Figure 4, A and B, indicates that large events were relatively slow. A plot of rise time versus amplitude (Fig. 4C) further demonstrates that large events tended to have longer T10-90s whereas many of the smallest events were among the fastest. The small fast sEPSCs (Fig. 4, A and B, trace 1) were likely to be action-potential-independent events originating at somatic and/or proximal dendritic sites, whereas the small slow sEPSCs (Fig. 4, A and B, trace 2) can be attributed to distal dendritic sites. The origin of the large events (Fig. 4, A and B, trace 3, and C) is more problematic. It is possible that some of the large and slow, presumably action-potential-dependent, events originated at distal synapses. However, segregation of the action-potential-dependent events to distal dendritic sites is unlikely. Alternately, action potential invasion of nerve terminals may cause multivesicular transmitter release. Transmitter release from these vesicles may not be perfectly synchronous. This is further supported by that fact that a notch was seen occasionally on the rising phase of large-amplitude events. The low frequency of clear notches on the rise phase of large-amplitude sEPSCs may be partly due to masking by noise and the fact that the delay between the release of vesicles is short. Therefore we suggest that asynchronous transmitter release may be a major mechanism underlying large slowly rising and decaying events (Vautrin and Barker 1995
). Higher transmitter concentration at the synaptic cleft, due to the release of large vesicles, may also contribute to larger events having a longer rise time and slower decay. Figure 4D shows that peak amplitude and average rate of rise were strongly correlated (r = 0.9 ± 0.07, 3 cells for sEPSCs and 4 cells for mEPSCs; the fastest rise rate observed was 350 pA/ms). This is expected because larger events represent the activity from greater amounts of transmitter and/or larger number of receptors.

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| FIG. 4.
Asynchronous transmitter release may contribute to the variability in the rise and decay of excitatory postsynaptic currents (EPSCs). A: averaged small fast (trace 1), small slow (trace 2), and large slow (trace 3) sEPSCs recorded in the absence of TTX. The trace superimposed with a doubleexponential fit is the average of all 448 events:T10-90 was 0.75 ms, f was 2.6 ms (90% of the total amplitude), and s was 16.3 ms. The small fast sEPSCs may be the action-potential-independent events originating at proximal sites. The small slow sEPSCs may be those originating at distal sites. Large slow events may be action potential dependent and generated at proximal sites. B: traces 1-3 scaled to the same peak amplitude. C: plot of rise time vs. amplitude showing that large, presumably action-potential-dependent events tended to have a slower rise time. D: average rise rate, calculated by dividing the peak amplitude by the T10-90, plotted as a function of peak amplitude. Linear regression analysis shows a strong correlation between the two parameters. In this cell Rs: ~10 M .
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| FIG. 5.
Inward rectification of AMPA-receptor-mediated EPSCs. A: records showing sEPSCs at 60 mV in the absence of TTX. Rs: 8 M . Large, presumably action-potential-dependent events were often seen. B: examples of sEPSCs at +60 mV. Large-amplitude events were absent, suggesting that sEPSCs were inwardly rectifying. C: superimposed averaged sEPSCs (all events) at +60 and 60 mV. At +60 mV, only relatively large events were detected such that the averaged sEPSC was larger than the ratio predicted. D: specimen records showing mEPSCs at 60 mV. Rs: 7 M . D, inset: averaged mEPSC. f = 2 ms; s = 8.8 ms. E: sections of recording at +60 mV. No mEPSCs could be reliably detected, suggesting that mEPSCs were also inwardly rectifying. A-C and D and E are from different layer I neurons.
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Rectification of AMPA-mediated EPSCs
In the presence of 20 µM D-APV, the amplitude of sEPSCs at
60 mV was up to 200 pA (Fig. 5A). Bath application of 10 µM CNQX eliminated all events, indicating that they were mediated by AMPA receptors. When the holding potential was changed to +60 mV, sEPSC amplitude was always <60 pA (Fig. 5B). These reversed sEPSCs were also blocked by 10 µM CNQX. The apparent frequency of the AMPA sEPSCs at +60 mV was less than half that at
60 mV (Fig. 5B). Similar results were seen in all layer I neurons tested (n = 8). These results indicate that the synaptic AMPA receptor channels on layer I neurons are inwardly rectifying. We estimated that the rectification ratio, as defined by the ratio of amplitudes of largest sEPSCs at
60 mV over those at +60 mV, was ~2-3. The ratio of averaged sEPSC amplitudes at
60 mV over that at +60 mV was <2 (Fig. 5C) because smaller events (5-10 pA) at +60 mV were not reliably detected or their waveform was so severely affected by noise that they were excluded from averaging.
The estimated rectification ratio implies that small-amplitude events, such as mEPSCs, may be buried in the noise at +60 mV, particularly because noise levels were often greater at positive potentials (compare the baseline noise in Fig. 5, D and E). At
60 mV, frequent mEPSCs with amplitudes between 10 and 30 pA were observed (Fig. 5D). On depolarization to +60 mV, clear mEPSCs were not apparent (Fig. 5E). Because the mean amplitude of mEPSCs was ~16 pA at
60 mV, the rectification ratio predicts that the mean mEPSC amplitude would be ~5 pA at +60 mV, near the noise level. Similar results were obtained in all layer I neurons tested (n = 8). In another five layer I neurons, in which the amplitude of many mEPSCs was >40 pA at
50 mV, clear mEPSCs were seen at +50 mV. The calculated rectification ratio was ~2. Further, the rise times of EPSCs at +50 mV were similar to those at
50 mV, suggesting that voltage control at each potential was similar.
NMDA EPSCs in layer I neurons
The inward rectification of AMPA EPSCs described above could reflect poor voltage control at the subsynaptic site. We reasoned that examination of current-voltage relations of the NMDA-receptor-mediated EPSCs might serve as an independent indicator of whether the observed inward rectification of AMPA EPSCs was due to space- and voltage-clamp problems. Additionally, it has been reported that the majority of hippocampal interneurons with inwardly rectifying AMPA mEPSCs is devoid of NMDA receptors (McBain and Dingledine 1993
). We tested whether neocortical layer I neurons also lack synaptic NMDA receptors.
sEPSCs recorded in the presence of 1.3 mM Mg2+ are shown in Fig. 6. At
60 mV, frequent fast sEPSCs with variable amplitudes were recorded (Fig. 6A). At a holding potential of +60 mV, frequent, variable-amplitude sEPSCs with much slower kinetics were observed (Fig. 6B). D-APV (20 µM) had little effect on the synaptic activity recorded at
60 mV, whereas slow sEPSCs at +60 mV were completely blocked (Fig. 6C). Further, D-APV also reduced the baseline "noise" at +60 mV (compare Fig. 6, B and C), suggesting tonic activation of NMDA receptors. Similar observations were obtained in all eight layer I neurons tested. In the presence of 0.5 µM TTX, D-APV-sensitive, slow NMDA mEPSCs were recorded at +60 mV in seven layer I neurons. In the absence of Mg2+ in the bathing solution, NMDA sEPSCs and mEPSCs were also recorded at
60 mV (Fig.6D). The decay of averaged NMDA sEPSCs was double exponential. At a holding potential of
60 mV,
f and
s were 31 ± 4.5 ms and 198 ± 30 ms, respectively (n = 5). At +60 mV,
f and
s were 61 ± 18 ms and 282 ± 45 ms, respectively (n = 4). These results indicate that layer I neurons possess synaptic NMDA receptor channels, consistent with previous studies of other mammalian central neurons (Bekkers and Stevens 1989
; Burgard and Hablitz 1993
; Nowak et al. 1984
; Stern et al. 1992
). The above results also suggest that reasonable voltage clamp was achieved in our experiments and that the observed inward rectification of AMPA-receptor-mediated sEPSCs and mEPSCs in layer I neurons was mostly due to the intrinsic conducting properties of these channels.

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| FIG. 6.
Layer I neurons have functional synaptic NMDA receptors. A: records showing predominantly AMPA-receptor-mediated sEPSCs with fast kinetics at 60 mV in the presence of 1.3 mM Mg2+. B: depolarization to +60 mV reveals NMDA sEPSCs. C: at +60 mV, bath application of 20 µM D-APV completely blocks slow NMDA sEPSCs. Note that a few small AMPA sEPSCs remain at +60 mV in the presence of D-APV. D: averaged NMDA sEPSCs at 60 mV (22 events) and +60 mV (19 events) in the presence of 10 µM CNQX and 0 mM Mg2+. Double-exponential fits are shown superimposed. Note the amplitude at +60 mV is larger than that at 60 mV.
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CTZ prolongs EPSCs
To further characterize the properties of EPSCs in layer I neurons, we examined the effects of 50 µM CTZ, a drugthat has been shown to slow the desensitization of AMPA, but not kainate, receptors in hippocampal neurons (Yamada and Tang 1993
). The most marked effect of CTZ was prolongation of sEPSC decays. No effect on the rate of rise of sEPSCs (Fig. 7) was observed. At
60 mV, CTZ slowed
f and
s of mEPSCs (n = 3) and sEPSCs (n = 4) from ~2.4 and 10 ms to ~4 and 20 ms, respectively. At +60 mV, CTZ slowed
f and
s of mEPSCs (n = 2) and sEPSCs (n = 3) from ~3.2 and 18 ms to ~5 and 35 ms, respectively. The amplitude of the fast component was increased ~20% by CTZ. The amplitude of the slow component was increased ~150% by CTZ for both mEPSCs and sEPSCs. CTZ slightly reduced the skew of the amplitude histogram of mEPSCs. The frequency of sEPSCS and mEPSCs in layer I neurons was increased by ~15%. The latter two effects were most likely due to the fact that CTZ increased the amplitude and thus the detection of small events, although a presynaptic effect of CTZ (Diamond and Jahr 1995
) cannot be ruled out. Averaged sEPSC amplitude at +60 mV was not increased, because the enhancement of amplitudes of individual sEPSCs was offset by the detection of more small events. Figure 7A also shows that the decay of the averaged sEPSC was slower at +60 mV (
f = 3 ms) than at
60 mV (
f = 2.2 ms; see also Fig. 5C). Similar voltage-dependent changes in decay have been reported for AMPA currents recorded in membrane patches (Barbour et al. 1994
; Raman and Trussell 1995).

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| FIG. 7.
Effects of cyclothiazide (CTZ) on EPSCs in layer I neurons. A: superimposed averaged sEPSCs at 60 and +60 mV in the absence of CTZ. In this cell, large events were infrequent such that the averaged sEPSC had a relatively small amplitude. Double-exponential fits are superimposed on the averaged sEPSCs with the parameters listed at right. Note the slower kinetics at +60 mV. B: bath application of 50 µM CTZ substantially prolonged the decay of the averaged sEPSCs at both 60 mV and +60 mV. Double-exponential functions were still needed to fit the decay. CTZ also increased the amplitude of sEPSCs at 60 mV. Increase in the amplitude of the averaged sEPSC at +60 mV was largely offset by the detection of more small events.
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DISCUSSION |
The main findings of this study are that rat neocortical layer I neurons display dual-component sEPSCs and mEPSCs. An AMPA-receptor-mediated component having rapid kinetics and substantial inwardly rectification dominates at RMP.
Rapid kinetics of AMPA EPSCs in layer I neurons
The present results show that, in rat neocortical layer I neurons, AMPA mEPSCs have very fast kinetics. T10-90s of averaged mEPSCs were ~0.3 ms and in some individual mEPSCs T10-90s were as fast as 0.15 ms. Decay time constants had a dominant component ~1.2 ms. If we take into account filtering by the cell-electrode circuit and the recording apparatus, T10-90 and
f may be near 0.2 and 1 ms, respectively, for AMPA-mediated mEPSCs in layer I neurons. These very rapid kinetics, combined with results from rapid application experiments (Jonas et al. 1994
; Koh et al. 1995
; Silver et al. 1996
), indicate that the synaptic glutamate pulse may be ~1 ms in duration. Given such a short pulse of glutamate, the decay of AMPA mEPSCs in layer I neurons would then be dominated by AMPA receptor deactivation kinetics. The kinetics of AMPA-receptor-mediated mEPSCs in neocortical layer I neurons, which are most like to be inhibitory neurons, were similar to those in excitatory cerebellar granule cells (Silver et al. 1996
), suggesting that EPSC kinetics cannot necessarily distinguish between excitatory and inhibitory cell types as suggested by Hestrin (1993)
.
The decay of averaged AMPA mEPSCs in most layer I neurons was best fitted by double-exponential functions with a dominant fast and small slow component. However, in every cell, some single-exponentially decaying events were found and, in a minority of cells, single-exponentially decaying events dominated such that averaged mEPSCs were well fitted by single-exponential functions. The origin of the small slow decay term, and why this term was absent in some EPSCs, is unclear. Several possible explanations are suggested by rapid glutamate application experiments. Spruston et al. (1995)
reported that, in membrane patches from hippocampal pyramidal cells, 1-ms applications of 1 mM glutamate induced responses with double-exponential deactivation kinetics, including a small slow term with a time constant of ~8 ms. The small slow component in our mEPSCs may be due to this slow deactivation. Alternatively, desensitization time constants with values of ~4 ms have been reported for AMPA receptors in several cell types (Jonas et al. 1994
; Koh et al. 1995
; Silver et al. 1996
). The small slow mEPSC component could therefore be partially due to desensitization. It has also been suggested that the clearance of glutamate from the synaptic cleft is biphasic with a small slow component (see Clements 1996
for review). This slow clearance could lead to AMPA receptor desensitization. The latter two possibilities are not mutually exclusive. The small slow component in the mEPSCs recorded here may be due to a combination of complex deactivation and partial desensitization. However, the variations in the small component in our mEPSCs suggest that difference in transmitter clearance resulting from different synaptic geometry may be more important. The fact that CTZ had a greater enhancing effect on the amplitude of the small slow component also suggests that the clearance of glutamate is sufficiently fast under normal conditions that desensitization of AMPA receptors contributes only slightly to EPSCs in layer I neurons. On being blocked by CTZ, desensitization and possibly rebinding (Mennerick and Zorumski 1995
) may result in an increase in the slowly decaying component.
Origin of the variability in sEPSC kinetics
The kinetics of mEPSCs in layer I neurons were variable. There was a positive correlation between rise and decay times, suggesting a contribution from dendritic filtering (Major 1993
; Rall 1977
; Spruston et al. 1993
). The modest r values suggest that dendritic filtering may not be linear (Spruston et al. 1993
) and may not be the only factor in shaping mEPSC waveform.
Another factor that may shape the waveform of mEPSCs is the timing of transmitter release (Vautrin and Barker 1995
). For single vesicle release, transmitter molecules in small vesicles are more likely to be released and bind to postsynaptic receptors in a short period of time. The opposite condition may occur with larger vesicles. Asynchronous release is more likely to happen under conditions of multiquantal release, such as occur during action potential-dependent release (Vautrin and Barker 1995
) and occasional large-amplitude miniature synaptic currents (Ulrich and Luscher 1993
). Further, it has been reported that a high glutamate concentration in the synaptic cleft resulting from a larger quantal release can produce a longer rise and decay time and a larger synaptic current (Barbour et al. 1994
; Mennerick and Zorumski 1995
; Trussell et al. 1993
). Our observations that many of the fastest events had the smallest amplitudes whereas many large events were relatively slower is consistent with the asynchronous release hypothesis, as is the finding that action-potential-dependent events were slower. The asynchrony of transmitter release and the concentration of transmitter in the synaptic cleft are likely to offset and/or mix with cable effects such that correlations among rise time, decay time, and mEPSC amplitude are attenuated or masked.
Origin of variability in mEPSC amplitudes
According to the quantal theory for synaptic transmission (Katz 1966
), mEPSCs represent spontaneous quantal events whose amplitudes are normally distributed. The distribution of mEPSCs amplitudes in layer I neurons was skewed toward large events. Several factors may contribute to this type of distribution, including variability in number of transmitter molecules in synaptic vesicles (Bekkers et al. 1990
), variability in postsynaptic receptor density (Edwards 1995
), occasional multiquantal release (Ulrich and Luscher 1993
), and changes in dendritic attenuation from synapse to synapse.
The peak amplitude of averaged layer I mEPSCs were ~16 pA at
60 mV. If we assume that average mEPSCs are due to the release of single, average-sized vesicles, then the quantal conductance of our mEPSCs in neocortical layer I neurons was 270 pS. This is in agreement with previous results in hippocampal pyramidal cells (Bekkers et al. 1990
; Jonas et al. 1993
), visual cortical interneurons (Stern et al. 1992
), and cerebellar granule cells (Silver et al. 1992
). The conductance of single AMPA channels in somatic membrane patches has been estimated to be ~25 pS for visual cortical nonpyramidal neurons (Hestrin 1993
) and hippocampal dentate basket cells (Koh et al. 1995
). If we assume that synaptic AMPA receptors on layer I neurons have a similar single-channel conductance, then on average, quantal events were generated by the opening of ~11 AMPA receptors.
Layer I neurons have functional NMDA receptors
NMDA-receptor-mediated sEPSCs and mEPSCs were recorded in all layer I neurons tested, indicating that these neurons possess synaptic NMDA receptors. At RMP, the decay of averaged NMDA sEPSCs and mEPSCs was best described by double-exponential functions with time constants similar to those reported for NMDA EPSCs in other neurons (Lester et al. 1990
; Perouansky and Yaari 1993
). In nominally Mg2+-free bathing solution, the NMDA component in layer I neurons demonstrated modest outward rectification and the decay was slower at +60 mV than at
60 mV, similar to reports in other neuronal types (Keller et al. 1991
; Konnerth et al. 1990
; Nowak et al. 1984
; Perouansky and Yaari 1993
).
Inward rectification of AMPA sEPSCs and mEPSCs
Cloned AMPA-type glutamate receptors lacking the GluR-2 subunit display inwardly rectifying current-voltage relations and are permeable to Ca2+ (see Hollmann and Heinemann 1994 for review). McBain and Dingledine (1993)
described strong inward rectification of AMPA mEPSCs in a subset of hippocampal interneurons. Combined patch-clamp and molecular studies have shown that a lack of GluR-2 mRNA results in inward rectification and high Ca2+ permeability in cultured hippocampal neurons (Bochet et al. 1994
). AMPA receptors on hippocampal basket cell somata show significant inward rectification and are highly permeable to Ca2+ (Koh et al. 1995
). This inward rectification might be due to binding of intracellular factors to the AMPA channels (Isa et al. 1995
; Kamboj et al. 1995
) or intrinsic voltage dependency in channel gating (Raman and Trussell 1995). In the present study, AMPA-receptor-mediated sEPSCs and mEPSCs, in all layer I neurons tested, showed substantial inward rectification. A recent immunostaining study indicates that a subpopulation of neocortical interneurons, including layer I neurons, lacks the GluR-2 subunit (Kharazia et al. 1996
). Therefore it is very likely that AMPA receptors on neocortical layer I neurons lack the GluR-2 subunit and are highly Ca2+ permeable. Such Ca2+ entry may provide local subsynaptic Ca2+ transients that could trigger a variety of Ca2+ signaling pathways.