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The Journal of Neuroscience, March 15, 2000, 20(6):2073-2085
Heterogeneous Conductance Levels of Native AMPA Receptors
T. Caitlin Smith1, Lu-Yang Wang2, and James R. Howe1, 2 1 Interdepartmental Neuroscience Program and 2 Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
The single-channel properties of AMPA receptors can affect information processing in neurons by influencing the amplitude and kinetics of synaptic currents, yet little is known about the unitary properties of native AMPA receptors in situ. Using whole-cell and outside-out patch-clamp recordings from granule cells in acute cerebellar slices, we found that migrating granule cells begin to express AMPA receptors before they arrive in the internal granule cell layer and receive synaptic input. At saturating agonist concentrations, the open probability of channels in outside-out patches from migrating cells was very high, allowing us to identify patches that contained only one or two active channels. Analysis of the single-channel activity in these patches showed that individual AMPA receptors exhibit as many as four distinguishable conductance levels. The conductance levels observed varied substantially for different channels, although on average the values fell within the range of unitary conductances estimated previously for synaptic AMPA receptors. In contrast to patches from migrating granule cells, we rarely observed directly resolvable single-channel currents in patches excised from the somata of granule cells in the internal granular layer, even though these cells gave large AMPA receptor whole-cell currents. We did, however, detect AMPA receptors with apparent unitary conductances of <1 pS in patches from both migrating and mature granule cells. Our results suggest that granule cells express a heterogeneous population of AMPA receptors, a subset of which are segregated to postsynaptic sites after synaptogenesis.
Key words: granule cell; cerebellum; AMPA receptor; glutamate; single channel; synaptic; extrasynaptic; development
INTRODUCTION
Although AMPA receptors mediate EPSCs in most CNS neurons, the single-channel properties of these receptors have not been directly studied in neurons in situ. AMPA receptors are multimeric assemblies of the glutamate receptor (GluR) subunits GluR1-4, and their subunit composition and stoichiometry, as well as RNA editing and alternative splicing, influence several important channel properties (Hollmann and Heinemann, 1994
; Bettler and Mulle, 1995
Studies of recombinant AMPA receptors have shown that homomeric, as well as heteromeric, channels display multiple conductance levels whose amplitudes depend on subunit composition (Swanson et al., 1997
Localization of synaptic AMPA receptors in situ at postsynaptic densities presents further barriers to studying channel properties directly. Thus information about the conductance of synaptic channels is limited to estimates obtained from nonstationary fluctuation analysis of synaptic currents. Recently, investigations of recombinant AMPA receptors demonstrated that the prevalence of sojourns in conductance substates varies with agonist concentration (Rosenmund et al., 1998
In the present study, we have recorded currents through single AMPA receptors at saturating agonist concentrations, under conditions at which the high popen of the channels enabled us to identify patches containing at most two active channels. We have taken advantage of the postnatal development of the cerebellum to record AMPA receptor currents at two developmental stages: (1) during migration through the molecular layer (ML) before the neurons receive synaptic input and (2) after synaptogenesis in the internal granular layer (IGL). We found that AMPA receptors expressed by migrating cerebellar granule cells exhibit as many as four conductance levels, which showed extensive heterogeneity from channel to channel. On average, larger-conductance channels in outside-out patches had conductances similar to those estimated for synaptic AMPA receptors at the mossy fiber-granule cell synapse (Traynelis et al., 1993
MATERIALS AND METHODS
Slice preparation and patch-clamp recordings. Mice of ages postnatal day 6 (P6)-P47 (C57-black-6; Charles River Laboratories, Wilmington, MA) were anesthetized with Metofane (Pitman-Moore) and decapitated, and the brains were removed in ice-cold oxygenated artificial CSF (ACSF). Parasagittal slices (150-200 µm thick) of cerebellum were cut with a vibratome. Slices were then incubated in ACSF at 37°C for 30-60 min and at room temperature (20-22°C) thereafter. ACSF contained (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 1.25 NaH2PO4H2O, 26 NaHCO3, 2 Na-pyruvate, 3 myo-inositol, and 0.5 ascorbic acid, pH 7.4, when oxygenated.
In the recording chamber, the slices were continuously perfused (~1-2 ml min
1) with ACSF that contained the potassium channel blockers tetraethylammonium chloride (10 mM) and 4-aminopyridine (0.1 mM) and the GABAA receptor antagonist bicuculline methylchloride (20 µM). Currents through NMDA receptors were blocked by addition of either 100 µM D-2-amino-5-phosphonovaleric acid (D-APV) or 20 µM D-APV and 20 µM 7-chlorokynurenic acid. All recordings were performed at room temperature. The pipette solution contained (in mM): 97.5 Cs-gluconate, 32.5 CsCl, 5 EGTA, 10 HEPES, 1 MgCl2, and 2 lidocaine n-ethyl bromide (QX314; to block currents through sodium channels), pH 7.2. To replace the diffusional loss of intracellular polyamines, 100 µM spermine was included in the pipette solution. Patch pipettes (7-12 M
) were made from borosilicate glass, coated with Sylgard (Dow Corning), and fire-polished. Granule cells were identified by their location, appearance, and small capacitance (1-4 pF). The developmental stages of granule cells were determined visually on the basis of their positions within the slices.
In outside-out patch recordings, cyclothiazide (100 µM) was added to the external solutions from a dimethylsulfoxide (DMSO) stock solution (final DMSO, 0.5%) to reduce AMPA receptor desensitization (Partin et al., 1993
Data acquisition and analysis. Whole-cell and single-channel currents were recorded using an EPC-9 patch-clamp amplifier. Data from whole-cell recordings were acquired and stored as described previously (Howe, 1996
= RC, exceeded 200 µsec. In most whole-cell recordings,
Outside-out patch recordings were low-pass filtered at 10 kHz (
3 dB; four-pole Bessel type) and stored on videotape. The replayed signals were redigitized at 94 kHz and were additionally low-pass filtered with a digital Gaussian filter. For spectral density analysis of steady-state agonist-evoked ensemble currents in outside-out patches, the data were digitally low-pass filtered at 2 kHz (
As described below, two independent methods of single-channel analysis were used to measure the amplitudes of single-channel currents. Both methods also gave estimates of the proportion of time spent at the various open levels. We measured a mean reversal potential of 0.85 ± 2.0 mV (n = 9 cells) in whole-cell recordings of AMPA-type currents, so we used a reversal potential of 0 mV to calculate single-channel conductances.
Mean low-variance analysis. To measure the amplitudes of completely resolved single-channel openings, we used the mean low-variance method of Patlak (1988)
Histograms of the low-variance open points were typically constructed using 75-100 bins, which were fitted with the sum of multiple Gaussian components using TAC software (Bruxton Corporation, Seattle, WA) to obtain mean currents for the multiple open levels in the records. Histograms of open points from longer portions of the record did not always show clear peaks; in such cases, we used the apparent open levels found during inspection of the data as initial values for the fit. All histograms were fitted with the minimum number of components required to give a good fit, with the restriction that the SD of each Gaussian component was no greater than twice the SD of the closed-channel current.
Hidden Markov analysis. To confirm our mean low-variance results, we also estimated open levels by hidden Markov analysis (Chung et al., 1990
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Models.
Model A is approximately based on the results of Rosenmund et al. (1998)
The initial values for rate constants between connected states were set to 1000 sec
Determining whether one or more channels were active in an outside-out patch. To test whether our presumed single-channel records indeed represented the activity of only one AMPA receptor, we compared the observed current levels in the record with those predicted from the binomial distribution if the observed channel activity were caused by several smaller channels instead of one channel with multiple open levels. An all-points histogram of the observed steady-state channel activity in an outside-out patch was plotted and fitted with one Gaussian component for the closed points and one Gaussian component for each of the open levels. From the multiple Gaussian fit to the results, the relative area of the Gaussian component corresponding to the closed points was used to calculate the open probability of an individual channel if the record arose from N identical channels (where N corresponds to the number of observed open levels). The popen was defined as:
This estimate of the popen of the hypothetical individual channels was used to calculate the probability P of finding j channels simultaneously open at a given time using the binomial relationship:
Results are given as the mean ± SEM.
![P<SUB>j</SUB>=(N!/[j!(N−j)!])(p<SUP>j</SUP><SUB><UP>open</UP></SUB>)(1−p<SUB><UP>open</UP></SUB>)<SUP>N−j</SUP>.](/content/vol20/issue6/fulltext/2073/img002.gif)
RESULTS
We observed previously that cerebellar granule cells begin to express functional AMPA receptors at approximately the time they leave the external germinal layer (EGL) in approximately the first postnatal week (Smith et al., 1999
Granule cells express functional AMPA receptors before synaptogenesis
Whole-cell currents from granule cells in the EGL, ML, and IGL were evoked by bath application of kainate, an agonist that produces sustained currents through AMPA-type channels because of incomplete receptor desensitization [Patneau et al. (1993)
In agreement with our previous results (Smith et al., 1999
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Figure 1. Whole-cell currents through AMPA receptors increase as cerebellar granule cells mature. The first four bars (left to right) denote mean currents evoked by saturating concentrations of kainate (300-600 µM) in EGL, ML, immature IGL, and mature IGL cells, respectively; the last bar on the right is the mean current evoked in mature IGL cells by 100 µM kainate. Inspection of all the data indicated that the steady-state current amplitudes were log-normally distributed. The logarithms of the individual current amplitudes were used to calculate the mean and SEM for each sample population. The figure shows the antilogs of the values (mean ± SEM) obtained from the log-normal distributions: EGL, 2.3 pA (n = 9 cells); ML, 27.5 pA (n = 5 cells); immature IGL (<P40), 57.5 pA (n = 9 cells); mature IGL (>P40), 178 pA (n = 3 cells); and mature IGL (100 µM kainate), 135 pA (n = 5 cells). There were no significant differences in whole-cell capacitance between the granule cells in the different groups.
Detectable AMPA receptor single-channel activity in outside-out patches from migrating granule cells
To study the properties of the channels underlying the AMPA-type whole-cell currents, we pulled outside-out patches from granule cells migrating through the ML and bath-applied saturating concentrations of kainate (1 mM) or glutamate (2 mM). Both agonists were applied in the presence of 100 µM cyclothiazide to reduce AMPA receptor desensitization (Partin et al., 1993
Although our whole-cell recordings indicated that kainate-type currents were minimal or absent under these experimental conditions (see above), the whole-cell results do not exclude the possibility that kainate receptors could have contributed to the observed single-channel activity during occasional escapes from desensitization. We believe, however, that our single-channel records were not contaminated with kainate receptor activity. First, although single-channel currents through kainate-type channels can be observed in patches from EGL granule cells, the very small conductances of the kainate receptors expressed in granule cells make detection of discrete single-channel currents unlikely at the developmental stages studied here (Smith et al., 1999
There were several characteristics common to the single-channel activity of the AMPA receptors that we studied in ML patches. At high agonist concentrations, all the channels displayed periods of high popen activity that lasted hundreds of milliseconds or even seconds (Fig. 2A). These high popen periods are reminiscent of the high popen activity seen with NMDA-type glutamate receptors (Jahr and Stevens, 1987
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Figure 2. Single-channel activity of an AMPA receptor expressed by a migrating granule cell. Holding potential,
The high popen behavior of the channels allowed us to identify patches that appeared to contain only one or two active channels. Almost all the channels (11 of 13) in these patches visited more than one conductance level, the only exceptions being channels with very small single-channel currents. Examples of transitions to conductance sublevels are shown in Figure 2B. Although almost all the data presented in this study were obtained at saturating agonist concentrations, we also recorded single-channel currents over a range of concentrations. Channels showing multiple open levels spent more of their open time in larger sublevels as the agonist concentration was increased, as reported previously for recombinant AMPA receptors (Rosenmund et al., 1998
Native AMPA receptors show up to four conductance sublevels
We limited our single-channel analysis to patches that appeared to contain only one or two active channels, as determined from analysis of the high popen behavior at 2 mM glutamate (with 100 µM cyclothiazide). In patches that appeared to contain two channels, there were often portions of the record in which only one channel was active, due to entry into long-lived closed or desensitized states. Several consistent characteristics of the records indicate that the multiple current levels that we observed are in fact conductance substates of single AMPA receptors, rather than currents through several smaller channels. First, there were apparently direct transitions between nonadjacent open levels. Second, for some channels, the open levels observed were clearly not multiples of each other, as would be expected if different open levels corresponded to the simultaneous opening of more than one channel. Third, the high popen periods contained many brief closings that appeared to be direct transitions from and to the largest open level. Finally, as noted above, the high popen periods were interrupted by shut periods that lasted up to hundreds of milliseconds. Given the long duration and relative infrequence of these shut periods, it is highly unlikely that two or more independently gating channels would give rise to these shut periods by opening and closing simultaneously. Therefore, if multiple channels contributed to the record, one would expect these long-lived closed periods to be bordered by periods in which only smaller current levels were observed. This was clearly not the case (see Fig. 2).
Although most of the larger-conductance channels that we studied displayed open levels that were not multiples of each other, the closely spaced open levels observed for smaller-conductance channels often appeared to be. To evaluate further the likelihood that the channel activity observed arose from multiple channels, we compared the observed proportion of time spent at each open level with the proportions predicted from the binomial distribution if the activity arose from multiple channels gating independently. For example, how likely was it that a patch appearing to contain only one active AMPA receptor with open levels of 3, 5, and 7 pS actually contained three identical channels of 2-3 pS? In Figure 3A, the filled bars illustrate the relative time one such channel spent at each open level at 2 mM glutamate; the open bars represent the predicted probabilities of observing one, two, or three independent channels open simultaneously (calculated as described in Materials and Methods). Clearly the results deviate from those predicted from the binomial distribution. A similar analysis was performed for eight different channels, and in each case the predicted proportion of time spent at each current level differed substantially from the proportions observed experimentally. We also calculated how much the area under the closed-points component (see Materials and Methods) would have to differ from its fitted area to give hypothetical popen values consistent with the observed results if the activity arose from multiple channels. In each case this difference exceeded 80% of the measured area of the closed-points component, which far exceeded the uncertainty of the measurements.
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Figure 3. The observed current levels in outside-out patch recordings are inconsistent with the activity of multiple channels. A, Analysis of data from a putative one-channel recording. The filled bars indicate the proportion of time at each open level (PN) observed during steady-state activity evoked by 2 mM glutamate (with cyclothiazide). The open bars indicate the proportion of time at each current level predicted from the binomial distribution (described in Materials and Methods) if the currents in the record arose from the activity of three independently gating channels. B, Mean ratios (± SEM) of the observed PN to the PN predicted from the binomial distribution (if the activity arose from multiple independently gating channels). The results are from eight records in which two (circles), three (squares), or four (triangles) open levels were evident (n = 2, 3, and 3 individual channels, respectively). Note that the y-axis is discontinuous. For points off the scale, the mean ratios are given in parentheses.
The mean results from this analysis of eight individual AMPA receptors are summarized in Figure 3B, which shows the average ratio of the observed proportion of time the record spent at a particular current level to the proportion predicted if the current levels arose from multiple channels. Because the predictions differ quite strikingly from the observed data, we believe that our presumed single-channel records are indeed likely to contain only one active channel, even when the larger open levels appear to be multiples of the smallest.
Granule cells express AMPA receptors with heterogeneous conductance levels
Although almost all the AMPA receptors that we studied in patches showed multiple open levels at saturating concentrations of agonist, there was considerable channel-to-channel variability in the amplitudes of the conductance sublevels, as well as in the number of levels detected. The single-channel currents through 13 channels were measured during activity evoked by 2 mM glutamate. Single-channel currents evoked by 1 mM kainate were also measured for 3 channels. Interestingly, although agonist-dependent differences in the apparent unitary conductance of AMPA-type channels have been found using fluctuation analysis (for example, see Jonas and Sakmann, 1992
Figure 4 illustrates examples of data from a smaller AMPA receptor (Fig. 4A,B) and a larger AMPA receptor (Fig. 4C, D) in outside-out patches from two different ML cells. Three small, closely spaced, open levels were detected for the AMPA receptor shown in Figure 4A. In Figure 4A the dotted lines show the currents for each open level superposed on examples of the activity of this channel. The three peaks in the histogram of low-variance open points (from a selected portion of the record) correspond to conductances of 3, 6, and 9 pS (Fig. 4B). The larger-conductance AMPA receptor in Figure 4C visited four detectable open levels. At 2 mM glutamate the channel spent most of its time at the largest conductance level (22 pS), but examples of long-lived sojourns at the two smallest levels (7 and 14 pS) were also evident. In addition, the record contained openings to a level with a mean conductance of 18 pS, most of which were short-lived. A histogram of low-variance open points obtained from this channel is shown in Figure 4D, with the four Gaussian fit to the results superposed.
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Figure 4. AMPA receptors expressed by migrating granule cells show heterogeneity in the number and amplitudes of their conductance sublevels. The zero current levels are indicated by solid lines. Open levels are indicated by dotted lines. Channel currents were activated by 2 mM glutamate in the presence of 100 µM cyclothiazide. A, Outside-out patch recording from a granule cell in the ML. Holding potential,
Some of the patch-to-patch variability in conductance levels may reflect differences in the repertoire of AMPA receptors expressed by different granule cells. It was clear, however, that individual cells expressed multiple types of AMPA receptors. In each patch that contained two active channels, the two channels showed distinguishably different conductance levels. An example of results from a patch that contained two channels is illustrated in Figure 5. At the beginning of the trace, only the relatively smaller channel is active. In the middle of the trace, both channels are active. Near the end of the trace in Figure 5A, the smaller channel is silent, leaving only the activity of the larger channel. Figure 5, B and C (traces taken from the beginning and end of the trace in Fig. 5A), illustrates that the channel in Figure 5Ba has a smaller single-channel conductance than does the one in Figure 5Bb. This is also evident when histograms of low-variance open points are overlaid (Fig. 5D; histograms were constructed from portions of the trace where only one channel was active).
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Figure 5. Two AMPA receptors with different single-channel conductances in the same outside-out patch. Holding potential,
We used two independent methods of single-channel analysis to estimate the open levels of 13 individual AMPA receptors in eight outside-out patches. Consideration of all the records indicated that 2-3 pS was the minimum separation from baseline, and between levels, that we could resolve (see Materials and Methods). At holding potentials of
We first used HMM to verify the amplitudes of open levels that we measured by mean low-variance analysis. The open levels estimated with HMM at three different filter settings (18.8, 9.4, and 4.7 kHz) were similar, and they agreed well with the values obtained from fits to the mean low-variance open points (data low-pass filtered at 2 kHz). The levels obtained with HMM and mean low-variance analysis were usually identical and, on average, agreed within 1 pS. The observation that the amplitudes of the open levels were relatively insensitive to low-pass filtering implies that the open levels detected are unlikely to reflect incompletely resolved transitions between different channel states. We then used HMM to estimate the open levels of channels not amenable to mean low-variance analysis because of their rapid kinetics. Although HMM is in principle able to detect very small signals, only levels above our resolution at 2 kHz filtering are depicted in Figure 6.
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Figure 6. Heterogeneous open levels of AMPA receptors expressed by granule cells in situ. Amplitudes of the open levels (in picosiemens) of 13 individual AMPA receptors observed in outside-out patch recordings (circles, squares, triangles, and diamonds show levels in order of decreasing amplitude). Current levels were measured at
Figure 6 summarizes the number and amplitudes of open levels we observed from the single-channel activity of 13 individual AMPA receptors. These channels exhibited from one to four detectable open levels that ranged in conductance from 3 to 44 pS. Interestingly, there is a correlation between the number of open levels detected and their amplitudes. It is possible that the smaller-conductance channels have additional open levels that are too small or too brief to detect. Alternatively, AMPA receptors may truly differ in the number of their open states.
In addition to providing another method to measure channel open levels, HMM allowed a quantitative assessment of the number and connectivity of open levels that best described the data. For the five largest conductance channels (Fig. 6, bracketed channels 9-13), HMM analysis showed that Model A with four open states (in order of increasing conductance) gave significantly better fits to the single-channel records than did analogous models having three open states. In addition, versions with five open states did not significantly improve the fit to the data. For three of five channels, models that allowed direct transitions between all open levels (Model B) gave better fits to the data than did Model A. For each channel, the worst fits were obtained using Model C, which disallowed direct transitions between open states. The results of the above comparisons are summarized in Table 1.
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Table 1. Results of hidden Markov comparisons for larger-conductance AMPA receptors The HMM analysis also confirmed our impression that different channels showed different kinetic behavior. Figure 7 shows the rate constants and mean open times obtained from fitting the four-open-state version of Model A to the activity of two different AMPA receptors (sampled at 23.5 kHz; filtered at 9.4 kHz). The mean open times for the channel with the relatively faster kinetics (Fig. 7B) are much briefer than the window duration used for mean low-variance analysis (330 µsec), and at 2 kHz low-pass filtering the record consisted primarily of incompletely resolved events. Although Model B usually gave better fits than Model A, the open times obtained with the two types of models were very similar for each channel analyzed. Thus although the connectivity of the states is somewhat unresolved, we believe that the mean open times estimated with HMM are good approximations of channel dwell times.
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Figure 7. Rate constants obtained from hidden Markov modeling for two channels in different ML patches. The mean dwell times (in milliseconds) are shown for the four open states (states 5-8) in bold type. Rate constants >1000 sec
Rectification behavior of AMPA receptors in granule cells in situ
One possible explanation for the heterogeneity we observed in single-channel conductances is that the channels in ML patches differ in subunit composition. Inclusion of the edited GluR2 subunit in coexpression experiments reduces the single-channel conductance of GluR4 channels (Swanson et al., 1997
We first asked whether granule cells show significant cell-to-cell variability in the average rectification behavior of AMPA receptors, as has been shown for granule cells in culture (Kamboj et al., 1995
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Figure 8. Whole-cell currents through AMPA receptors in granule cells show linear or outwardly rectifying current-voltage relations, as do some ensemble patch currents. A, B, Whole-cell I-V curves from a granule cell in the ML (A) and the IGL (B). C, I-V curve (a) and ensemble currents (b) from an outside-out patch excised from a granule cell in the ML. Several channels were active in this patch. Holding potentials (in millivolts) are indicated to the left of the traces in b. Data were sampled at 31.3 kHz and low-pass filtered at 2 kHz (
In contrast to the whole-cell results, agonist-evoked ensemble currents in outside-out patches from migrating granule cells showed both inward and outward rectification, suggesting that these patches contained different subsets of AMPA receptors. In four patches, we observed linear or outwardly rectifying I-V relations (Fig. 8C), whereas three other patches showed inward rectification. These results suggest that the linear I-V relations of whole-cell currents may reflect the average behavior of a heterogeneous population of AMPA receptors, rather than reflecting the rectification behavior of a single type of AMPA receptor.
The heterogeneous rectification behavior of AMPA receptors was also evident in single-channel recordings. Some channels exhibited rectification behavior similar to that in the multichannel patch shown in Figure 8C, which passed outward currents at positive potentials, whereas others showed some flickery channel block, as if the channels might be weakly blocked by internal polyamines. Still other channels, such as the one illustrated in Figure 9, appeared to be more strongly blocked by internal polyamines, exhibiting a marked decrease in popen at positive potentials. This channel showed long silent periods at positive potentials that were occasionally interrupted by shorter periods of activity in which little or no flickery channel block was evident (Fig. 9A, top two traces). Interestingly, the single-channel I-V curve exhibited only slight inward rectification (Fig. 9B, filled circles), whereas the mean current measured for representative portions of the record at each potential showed strong inward rectification (open circles). Comparison of the two I-V curves in Figure 9B demonstrates that the rectification behavior of this channel is manifest primarily as a dramatic reduction in popen at positive potentials. Taken together, these data indicate that migrating granule cells express AMPA receptors that show substantial heterogeneity in both their single-channel conductances and rectification.
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Figure 9. Inwardly rectifying AMPA receptor expressed by a migrating granule cell. A, Outside-out patch recording from a granule cell in the ML. Holding potentials (in millivolts) are indicated to the left of the traces. Glutamate (2 mM) and cyclothiazide (100 µM) were present throughout the recording. Data were sampled at 31.3 kHz and low-pass filtered at 2 kHz (
Granule cells express femtosiemens-conductance AMPA receptors
Consistent with the size of the single-channel currents described above, most multichannel patches from ML cells showed large increases in current noise after application of agonist (Fig. 10A). In contrast, IGL patches that gave agonist-evoked currents had much smaller increases in current noise (Fig. 10B), and directly resolvable single-channel currents were noticeably absent in IGL patches with one exception. In addition, the frequency with which agonist-evoked currents were observed was lower in IGL patches (5 of 10) than in ML patches (28 of 31).
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Figure 10. Granule cells express AMPA receptors with femtoseiman conductances. A, Top, Outside-out patch from a granule cell in the ML. Data were sampled at 9.4 kHz and low-pass filtered at 2 kHz ( noise = 3.7 pS (not corrected for popen). B, Top, Outside-out patch from a mature IGL cell (P47). Note the much smaller agonist-induced current noise compared with the recording in A. Data were sampled at 9.4 kHz and low-pass filtered at 2 kHz (
Our results suggest that large-conductance AMPA receptors are primarily absent from the cell bodies of IGL granule cells, although smaller-conductance channels are still somatically localized. In each of the five IGL patches that responded to agonist, we observed small inward currents that developed and recovered gradually (Fig. 10B). Similar currents were observed in two ML patches. We believe that these low-noise responses are mediated by AMPA rather than by kainate receptors, because currents evoked by kainate (in the absence of concanavalin A) are blocked by the AMPA receptor antagonist GYKI 53655 (Smith et al., 1999
Taken together, our results suggest that granule cells in situ express at least two populations of AMPA receptors on their cell bodies during development: larger-conductance AMPA receptors expressed by migrating cells that become relatively inaccessible to a patch pipette after reaching the IGL and femtosiemens-conductance AMPA receptors that are somatically expressed by granule cells in the ML and IGL.
DISCUSSION
One of our main findings is that granule cells in situ express AMPA receptors with markedly different conductances. The femtosiemens-conductance channels we detected in some patches are similar to channels in cultured granule cells (Cull-Candy et al., 1988
AMPA receptors expressed in cerebellar granule cells in situ
Our results suggest that different populations of AMPA receptors may have different subcellular distributions in mature granule cells. The observation that the larger-conductance AMPA receptors (largest open level > 2 pS) became inaccessible to a patch pipette soon after granule cells arrive in the IGL suggests that these channels cluster at synapses. In contrast, the presence of femtosiemens-conductance channels in patches excised from IGL cell bodies indicates that they are located extrasynaptically, although we cannot exclude the possibility that they are also expressed at synapses. Our finding that the density of larger-conductance AMPA receptors is low on IGL cell bodies is consistent with the results of Silver et al. (1996a)
The conductance of the femtosiemens channels agrees well with the estimated conductance of GluR2 homomers and is smaller than that of receptors comprised of significant amounts of GluR1, GluR3, or GluR4 (Swanson et al., 1997
Although we do not know whether expression of the larger-conductance channels present in ML cells continues in IGL cells, synaptic clustering seems plausible, because the apparent lack of somatic expression in IGL cells coincides with synaptogenesis. The whole-cell
Heterogeneity of AMPA receptor single-channel properties
Another main finding of this work is that the larger-conductance AMPA receptors expressed by migrating granule cells exhibit considerable heterogeneity in their conductances, probably reflecting differences in subunit composition and perhaps differences in the relative contribution of alternative splice variants. Granule cells express mainly GluR2, GluR4, GluR4c, and to a lesser extent GluR1 (Monyer et al., 1991
Several channels that we studied showed four distinguishable open levels. Invariably, channels showing fewer conductance levels were those where the detection of conductance sublevels would have been limited by the small size of the single-channel currents. Thus the smaller channels may have had additional sublevels that were below our resolution. Rosenmund et al. (1998)
Functional implications of heterogeneous AMPA receptor properties
Whether the AMPA receptors expressed by migrating granule cells serve a developmental function is unknown. A direct role in migration seems unlikely because of the observation that the AMPA receptor antagonist CNQX had no effect on granule cell migration (Komuro and Rakic, 1993
It is also possible that the appearance of AMPA receptors in noninnervated granule cells simply reflects the preparation for impending synaptogenesis. If the channels in migrating cells are destined for synapses, this raises the possibility that the synaptic population of AMPA receptors is heterogeneous. Synaptic currents at the mossy fiber-granule cell synapse show approximately linear I-V relations (Silver et al., 1996b
Although individual neurons can selectively target subsets of AMPA receptors to different synapses (Rubio and Wenthold, 1997
