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NEUROSCIENCE |
1 Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK
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Abstract |
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) from
= 6.9, to 11.2 pS, when glutamate was increased from 10 µM to 10 mM. The kinetic behaviour of AMPAR channels was also influenced by agonist concentration, with an increase in ‘bursty’ events at higher concentrations. In contrast, kainate channels were characterized by brief openings without bursts. Consistent with the view that ‘bursty’ events arose from AMPARs, these openings decreased in the presence of the AMPAR blocker GYKI 53655. Furthermore, our experiments revealed a concentration-dependent increase in the number of conductance states during an individual AMPAR opening; AMPAR channels displayed up to four distinct levels. Our results are consistent with the view that the AMPAR channel conductance depends on the number of transmitter molecules bound in CA1 cells. We consider the implications of these findings for the change in EPSC properties during long-term potentiation (LTP).
(Received 30 November 2005;
accepted after revision 28 February 2006;
first published online 9 March 2006)
Corresponding author S. G. Cull-Candy: Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK. Email: s.cull-candy{at}ucl.ac.uk
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Introduction |
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A great deal is known about subunit expression (Ritter et al. 2002) and macroscopic characteristics of non-NMDA receptors in hippocampal CA1 cells (Jonas & Sakmann, 1992; Spruston et al. 1995; Bureau et al. 1999; Banke et al. 2000; Benke et al. 2001; Shi et al. 2001; Andrasfalvy et al. 2003). However, the single-channel properties of AMPA and kainate receptors have received much less attention. Because of this, it has been difficult to relate functional changes in the synaptic current with single-channel behaviour in these cells. For example, although the synaptic channel conductance has been estimated (Benke et al. 1998; Benke et al. 2001), it is unclear whether the values obtained match the directly observed single-channel openings. This is a significant consideration since such openings could arise from a homogeneous population of receptor channels, or a highly mixed population. The presence of a mixed channel population (Cull-Candy et al. 1988; Swanson et al. 1996) could profoundly influence any estimate of channel conductance, and complicate the interpretation of the increased channel conductance observed during LTP induction. For example, downregulation of a low-conductance channel within a mixed population, would yield an apparent increase in weighted mean channel conductance estimated from non-stationary noise analysis. From previous studies on individual AMPA and kainate receptor channels in other systems, it was expected that non-NMDARs would display fast channel kinetics with small multiple conductance openings between 0.2 and 25 pS, depending on their subunit composition (Cull-Candy & Usowicz, 1987; Jahr & Stevens, 1987; Swanson et al. 1996; Swanson et al. 1997; Banke et al. 2000; Smith & Howe, 2000; Jin et al. 2003; Oh & Derkach, 2005).
While the majority of fast synaptic transmission in the CNS is mediated by AMPARs, kainate receptors can contribute to excitatory postsynaptic currents at certain synapses, and can also participate in modulating synaptic transmission through their presence in the nerve terminal (Frerking & Nicoll, 2000; Lerma et al. 2001). In the present experiments it was also of interest, and necessary, to characterize events arising from kainate receptors, to allow an unequivocal distinction between different non-NMDAR channels.
AMPA and kainate receptors are often composed of heteromeric assemblies of subunits arising from multiple genes. Hippocampal CA1 pyramidal neurones are known to express mRNA for all four AMPAR subunits (Hollmann & Heinemann, 1994; Monyer et al. 1999). Furthermore, both flip and flop subunit variants are represented in CA1 pyramidal cells, although flop isoforms may tend to predominate at the age we have examined (Bahn & Wisden et al. 2000). Potentially, this could give rise to a considerable variety of AMPAR subtypes. However, at this age the two main AMPAR populations in these cells are likely to be composed of GluR1/GluR2 and GluR2/GluR3 subunit assemblies (Wenthold et al. 1996). Further, it has been demonstrated that GluR6 and KA2 kainate receptor subunits are also expressed in these cells; if others are present they occur at very low level (Wisden & Seeburg, 1993b; Bettler et al. 1992; Herb et al. 1992; Bureau et al. 1999). Kainate receptors in CA1 pyramidal cells could therefore function as homomeric GluR6 assemblies, which would be activated by kainate but not AMPA (see Herb et al. 1992) – or as a heteromeric combination of GluR6/KA2, which would respond to both agonists. The fast component of the EPSC in these cells appears to be mediated entirely by AMPARs, with no contribution from postsynaptic kainate receptors (Frerking et al. 1998). Furthermore, previous studies on macroscopic receptor currents have found that glutamate application onto membrane patches of CA1 cells activates predominantly AMPARs (Spruston et al. 1995).
Here we have compared functional properties of single AMPA and kainate receptor channels, and demonstrated that their conductance and kinetic properties are agonist concentration dependent in hippocampal CA1 pyramidal neurones. These findings have implications for the interpretation of changes in EPSC properties associated with LTP induction.
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Methods |
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For slicing, we used an ice-cold Ringer solution with the following composition (mM): 125 NaCl, 2.5 KCl, 1 CaCl2 2 MgCl2, 25 NaHCO3, 1.25 NaH2PO4, 25 glucose, 1 kynurenic acid; pH 7.4. The recording solution contained (mM) 125 NaCl, 3 KCl, 1 CaCl2 2 MgCl2, NaHCO3, glucose, 1 µM tetrodotoxin (TTX; Sigma), 10 µM SR95531 (Tocris Cookson, Bristol), 2 µM strychnine (Sigma, Poole, UK), 2 CsCl; pH 7.4. Furthermore, the NMDA receptor antagonists AP5 (50 µM) and 7-chlorokynurenic acid (20 µM) were added to the external recording solution. In some experiments the AMPAR antagonists GYKI 53655 (100 µM), SYM 2206 (100 µM) or NBQX (10–30 µM) were added to the external solution. Furthermore channels were activated by bath application of glutamate, AMPA, kainate or SYM 2081 (all from Tocris Cookson, Bristol, UK) at concentrations indicated. We did not attempt to compensate for changes in osmolarity or ion concentration produced by bath application of agonist solutions. Patch pipettes were filled with an intracellular solution containing (mM): 125 CsCl, 10 Hepes, 10 BAPTA, 10 TEACl, 1 N-(2,6-dimethylphenylcarbamoylmethyl) triethyl ammonium bromide (QX314), 2 Na2ATP, 2 MgATP, 0.3 Na3GTP, and 0.5 CaCl2, adjusted to pH 7.25 with CsOH, giving a final osmolarity of 285 ± 5 mOsmol/l.
Slice preparation and patch-clamp recording procedures
Preparation of hippocampal slices was carried out in accordance with the UK Animals (Scientific Procedures) Act of 1986. After decapitation of 12-day-old-rats (P12), their brains were rapidly removed as previously described (Misra et al. 2000). Horizontal slices (250 µm) were cut using a vibrating microslicer (DTK-1000; Dosaka EM Co., Kyoto, Japan). The slice contained the ventral hippocampus, entorhinal, perirhinal, and temporal cortices. Several slices were then maintained in a holding chamber containing slicing solution saturated with 95% O2–5% CO2, pH 7.4 and kept at room temperature for at least 1 h. For electrophysiological examination, these were transferred to a recording chamber perfused continuously with bubbled external solution, and viewed on an Axioskop-FS microscope (Zeiss, Welwyn Garden City, UK).
Patch-pipettes were pulled from thick-walled borosilicate glass capillaries (GC-150F; Harvard Apparatus Ltd, Edenbridge, UK), coated with Sylgard resin (Dow Corning, USA) and fire polished to a final resistance of 10–12 M
when filled with pipette solution. Outside-out patches were excised from visually identified pyramidal neurones in the CA1 layer. Before recordings were initiated, the patch-electrode noise level was checked. A root mean square noise level of <0.3 pA (bandwidth 5 kHz) was considered acceptable.
The actual range we obtained was 0.18–0.3 pA; to further minimize the noise for analysis, data were filtered at 1–2 kHz.
Before patches were exposed to bath-applied agonists, control recordings were made. Patches that showed any spontaneous channel activity (about 20% of all patches) were discarded. All channels examined were activated by steady bath application of agonist.
Steady-state single-channel activity was recorded at room temperature using a Axopatch 200A clamp amplifier (Axon Instruments, Union City, CA) and stored on digital audio tape (DC –20 kHz; DTR-1204; BioLogic, Claix, France.
Data acquisition and analysis
For analysis, single-channel currents were replayed from tape, amplified, filtered at 1–2 kHz (four-pole Bessel type) and digitized at 20 kHz using a CED 1401 Interface (Cambridge Electronic design, Cambridge, UK). Each digitized record was analysed using SCAN, an interactive computer program (http://www.ucl.ac.uk/Pharmacology/dc.html) that fits the time-course of each individual event with the step response of the recording system (Colquhoun & Sigworth, 1995). Distributions of amplitudes, and open and shut times were obtained from the time-course-fitted data.
Amplitude histograms were fitted with multiple Gaussian components, until an optimal fit was obtained; depending on the patch, this number varied between three and five. The amplitude histograms were obtained from fitted amplitudes (SCAN) and fitted with a mixture of Gaussians. Only openings longer than 1 ms (
2.5 x filter rise time) were included. Amplitude histograms were initially fitted with three Gaussians; additional components (5–6) were added, provided the fit showed a marked improvement. If an additional Gaussian did not further improve the fit, they were removed. To compare amplitude distributions at different glutamate concentrations, we calculated the mean of all amplitudes, in all patches exposed to the same concentration.
Distributions of shut times, open times and burst lengths were made using a logarithmic transformation of the abscissa and a square root transformation of the ordinate. The resolution of open and shut times was set to 80–140 µs. In our histograms the frequency tends to fall off within this time window, and events briefer than this were not detected. We did not correct for these missed events, since our analysis was focused mainly on events >1 ms. In those patches where we estimated the fraction of events <100 µs (the most frequently used resolution), this varied betweeen 1% and 22%. Distributions were fitted with probability density functions that were a mixture of two to four exponential components. Bursts of openings were defined as individual openings separated by shut times of duration less than a critical shut time, 
crit. The crit values were calculated so that the number of long shut times that were misclassified as within-bursts was equal to the number of short shut times that were misclassified as between-bursts by numerical solution of the equation:
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fast and slow are the two shortest exponentials obtained from the shut time distribution. Higher-order activation clusters were not analysed. |
Results |
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We were interested to determine basic properties of non-NMDAR channels activated by the transmitter (glutamate) over a broad range of concentrations, including those attained in the cleft during the transmission process (1–5 mM; Clements, 1996; Bergles et al. 1999; Barbour, 2001). To activate all non-NMDAR subtypes present we initially applied a high concentration (10 or 20 mM) of glutamate to outside-out patches of CA1 pyramidal cells in thin hippocampal slices (in the presence of AP5 and 7-chlorokynurenic acid, to block NMDARs). However, to verify, separately, that kainate receptors could be detected, patches were also exposed to the selective kainate agonist SYM 2081.
Figure 1 shows representative responses from outside-out patches (at membrane potential, Vm = –100 mV), exposed to 10 mM glutamate (Fig. 1A and B), and 100 µM SYM 2081 (Fig. 1C and D). SYM 2081 is thought be 1500-fold more potent for kainate receptors than AMPAR (Donevan et al. 1998). While no channel activity was detectable in the absence of these agonists, inward currents were clearly activated during drug application. Figure 1B and D illustrates single-channel records (same patches as in A and C), on a faster time scale – to permit individual events to be resolved.
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Single-channel currents activated by increasing concentrations of glutamate
We next tested whether the properties of single channels activated by glutamate were influenced by transmitter concentration (Rosenmund et al. 1998; Smith & Howe, 2000), as this could have implications for the interpretation of a change in channel conductance associated with LTP in CA1 cells (Benke et al. 1998). Figure 2A shows channel activity in response to three different glutamate concentrations (Vm = –100 mV). We obtained a low frequency of brief openings in the presence of 200 nM glutamate (Fig. 2A, left traces). On the other hand, from a cursory examination it is apparent that at higher glutamate concentrations (10 µM or 20 mM), the frequency, mean duration, and mean amplitude of openings, all increased dramatically. Indeed, because of the long duration of events seen at the highest concentration of glutamate (20 mM), many openings displayed exceptionally well-resolved conductance levels (see Fig. 2A, right traces).
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When we examined data from all of the patches that were exposed to the higher concentrations of glutamate (10 µM and 20 mM), it was possible to identify seven main conductance levels (see below). While the number present varied from patch to patch (between four and six), the fitted Gaussians invariably fell into one of these identified levels. This is not unexpected for cells where the population of native receptors may well show heterogeneity.
Our mean single-channel conductance estimates (mean ±
S.E.M.) were, in 200 nM glutamate
= 5.0 ± 0.03 pS (n
= 4 patches); in 10 µM glutamate
= 6.9 ± 0.2 pS (n
= 5); and in 20 mM glutamate
= 11.2 ± 0.2 pS (n
= 6). These differences are statistically significant between all three values (Student's t test, P < 0.01). Thus, in all patches studied, we found an approximate doubling in mean single-channel conductance when glutamate concentration was increased from 10 µM to 20 mM.
In addition, from examining the single-channel openings, there appeared to be a clear change in their kinetic properties with increasing glutamate concentration (Fig. 3A). We investigated this by constructing histograms of shut times and open periods (defined in Methods). The distributions of shut times, such as those shown in Fig. 3A, were best fitted with three or four exponential components when channels were activated by 200 nM, 10 µM and 20 mM glutamate.
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1
= 0.34 ± 0.08 ms (8 ± 1%), 2
= 7.0 ± 0.7 ms (44 ± 3%), 3
= 30.2 ± 2 ms (10 ± 1%) and 4
= 182 ± 4 ms (38 ± 2%) (n
= 4 patches); in 10 µM glutamate, 1
= 0.18 ± 0.01 ms (14 ± 2%), 2
= 8.5 ± 0.9 ms (72 ± 5%) and 3
= 37.5 ± 2.2 ms (14 ± 3%) (n
= 7); and in 20 mM glutamate, 1
= 0.14 ± 0.01 ms (34 ± 2%), 2
= 2.7 ± 0.2 (34 ± 3%), 3
= 28.6 ± 4.9 (19 ± 2%) and 4
= 345 ± 50 (13 ± 1%) (n
= 9).
In contrast with 1 and 4, there was no clear evidence that the time constants 2 and 3, or their relative areas, were dependent on glutamate concentration. Whereas 2, 3 and 4 are difficult to interpret, as they are influenced by factors such as the number of channels within a patch (which is unknown, and could vary over a wide range), 1 is thought to reflect the shut time within a burst. This value should not therefore be affected by the number of channels activated. While 1 itself showed no significant dependence on glutamate concentration, its relative area showed a clear increase. We obtained a statistically significant difference between 10 µM and 20 mM glutamate (Student's t test, P < 0.01). This reflects the fact that at higher glutamate concentrations, channel openings became more ‘bursty’ (see below).
As illustrated in Fig. 3B, an adequate description of the open periods required distributions to be fitted with the sum of two exponential components in the presence of 200 nM glutamate, and three exponentials in the presence of 10 µM and 20 mM glutamate. As the open period measured the total time that a channel was open, the value obtained was independent of the conductance level(s) adopted during the opening. The mean time constants (and relative areas) were estimated to be: in 200 nM glutamate, 1
= 0.23 ± 0.02 ms (75 ± 2%), and 2
= 1.32 ± 0.05 ms (25 ± 2%) (n
= 4); in 10 µM glutamate, 1
= 0.29 ± 0.01 (41 ± 2%), 2
= 1.14 ± 0.04 (38 ± 3%), and 3
= 3.7 ± 0.2 (21 ± 3%) (n
= 7); and in 20 mM glutamate, 1
= 0.15 ± 0.01 (37 ± 2%), 2
= 2.1 ± 0.2 (35 ± 4%) and 3
= 5.8 ± 0.7 (28 ± 5%) (n
= 9).
The mean value of the fastest component (1) was not significantly affected by glutamate concentration, whereas its relative area was significantly decreased in the presence of higher glutamate concentrations (10 µM or 20 mM) compared with that found in 200 nM glutamate (Student's t test, P < 0.05). Furthermore, in none of the four patches exposed to 200 nM glutamate could we detect the longest open period component (3). At 20 mM glutamate the exponentials 2 and 3 were larger than in 10 µM glutamate; the difference was statistically significant only for 2 (Student's t test, P < 0.05). Visually identified superimposed openings were not included in the analysis. Exclusion of such ‘double’ events, which are more frequent at longer open times, will mean that 3 and its corresponding area will be slightly underestimated at the higher glutamate concentrations. Hence, the real concentration dependence of open times will be greater than observed. As is apparent from Fig. 3B, the overall effect of increasing glutamate concentration, was to shift the open periods to the right.
Burst length distributions (Fig. 4A) were generally fitted with the sum of three exponentials. To decide whether or not the brief closings were occurring within a burst, we estimated a critical shut time (crit) for each patch (see Methods). The mean values obtained for crit were: 0.37 ± 0.03 ms in 10 µM glutamate, and 0.33 ± 0.04 ms in 20 mM glutamate. Because of the relatively small number of bursts (and hence the small area of 1) in the presence of 200 nM glutamate, we did not attempt to estimate burst length at this concentration. The burst length parameters could, however, be estimated for 10 µM, and 20 mM glutamate. These were: in 10 µM glutamate, 1
= 0.22 ± 0.02 ms (42 ± 2), 2
= 1.3 ± 0.07 ms (44 ± 1) and 3
= 5.5 ± 0.4 ms (14 ± 2) (n
= 7); and in 20 mM glutamate, 1
= 0.24 ± 0.04 ms (40 ± 2), 2
= 2.9 ± 0.4 ms (31 ± 3) and 3
= 9.1 ± 0.6 ms (29 ± 4) (n
= 9). While 1 and its relative area were similar at both glutamate concentrations (10 µM and 20 mM), 2 and 3 appeared to increase with concentration, and the relative area for 3 was larger in 20 mM glutamate. The increase in 3 was statistically significant (Student's t test, P < 0.05).
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1 of the shut time histograms that we observed in 20 mM glutamate. A summary of the kinetic description for the different agonists is given in Table 1 (open periods), Table 2 (shut times) and Table 3 (burst lengths). It is worth noting that the increase in burst length and open period that we observed with increasing glutamate concentration, would suggest that an alteration in cleft glutamate concentration profile may be capable of influencing EPSC time-course by modifying channel kinetics.
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1 reflected predominantly kainate receptor activations. Indeed, previous studies have suggested that glutamate activates predominantly AMPARs in these cells (see Spruston et al. 1995). Single-channel currents activated by increasing concentrations of AMPA
We next considered whether single-channel currents exhibited concentration dependence when activated by other agonists. Again, we examined amplitudes, shut times, open periods and burst behaviour of individual openings activated by 1 µM and 10 µM AMPA. Figure 5 shows recordings obtained from a patch exposed to 1 µM and 10 µM AMPA, and the corresponding amplitude histograms fitted with multiple Gaussians (five in 1 µM, and three in 10 µM AMPA). As we previously observed with glutamate, when data from all patches were examined, it was possible to identify seven main conductance levels (see below).
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= 6.1 pS in 1 µM AMPA; and
= 9.3 pS in 10 µM AMPA. Overall, the mean conductance was:
= 6.3 ± 0.01 pS (n
= 7cells) in 1 µM AMPA; and
= 7.7 ± 0.07 pS (n
= 5 cells) in 10 µM AMPA. Although the mean increase in amplitude was not statistically significant across all patches, the effect appeared consistent. It was expected that with higher concentrations of AMPA the increase would have been apparent. However, in all such patches, the intense level of channel activity produced by high AMPA concentrations precluded detailed analysis.
Typical shut time distributions for the different AMPA concentrations are shown in Fig. 6A. The histograms were fitted with three exponential components. The mean time constants (and relative areas) in 1 µM AMPA were, 1
= 0.22 ± 0.02 (19 ± 4%), 2
= 9.2 ± 0.3 (46 ± 2%), 3
= 93 ± 13 ms (35 ± 2%) (n
= 7). In 10 µM AMPA the values were, 1
= 0.16 ± 0.07 (23 ± 1%), 2
= 8.5 ± 0.3 (67 ± 1%) and 3
= 55.7 ± 7.5 (10 ± 1%) (n
= 6). The shorter shut time components (1 and 2), and their relative areas, were similar at the two concentrations. The slowest component (3) and its relative area appeared smaller in 10 µM AMPA. This difference was statistically significant for the relative area of 3 (Student's t test, P < 0.05). In contrast with our glutamate data, a fourth exponential was not detected in any of the patches exposed to AMPA.
At both AMPA concentrations, the open period histograms (Fig. 6B) could be satisfactorily fitted with three exponentials in all patches. The mean time constants (and relative areas) obtained with 1 µM AMPA were, 1
= 0.2 ± 0.01 (56 ± 2%), 2
= 1.02 ± 0.04 (33 ± 1%), 3
= 2.9 ± 0.1 ms (11 ± 1%) (n
= 7). In 10 µM AMPA the values were, 1
= 0.24 ± 0.02 (31 ± 2%), 2
= 2.1 ± 0.1 (49 ± 3%) and 3
= 5.8 ± 0.2 (20 ± 5%) (n
= 6). While the fastest component (1) showed no significant change with concentration, its relative area was significantly smaller at the higher concentration (10 µM AMPA). On the other hand, both 2 and 3 were significantly smaller in the lower AMPA concentration (1 µM). The relative area of 2 was significantly reduced in 1 µM AMPA, whereas the relative area of 3 showed no significant change.
As illustrated in Fig. 7A, at both AMPA concentrations the burst length distributions were best fitted with the sum of three exponential components. The burst length parameters were, in 1 µM AMPA, 1
= 0.19 ± 0.02 ms (51 ± 1%), 2
= 1.08 ± 0.06 ms (36 ± 1%) and 3
= 3.94 ± 0.02 ms (13 ± 1%) (n
= 7). In 10 µM AMPA the values were 1
= 0.22 ± 0.04 (38 ± 4%), 2
= 2.1 ± 0.3 (39 ± 3%), and 3
= 9.2 ± 1.6 (23 ± 3%) (n
= 6). Whereas the fastest component (1) showed no significant concentration dependence, both 2 and 3 were significantly increased in 10 µM AMPA. The relative areas of all three time constants showed no significant change. The mean number of openings per burst (1.26 ± 0.01 at 1 µM AMPA and 1.29 ± 0.02 at 10 µM AMPA, Fig. 7B) was similar at these two AMPA concentrations examined.
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Single-channel currents activated by kainate and SYM 2081
To test further our working hypothesis, that brief events in these patches arose selectively from native kainate receptors in CA1 cells, we examined channels activated by kainate and SYM 2081 (selective kainate agonist) (Fig. 8A and C). In all patches examined, 30 µM kainate activated a high frequency of very brief events. At higher kainate concentrations (100 µM) individual openings could no longer be clearly resolved (data not shown). The kainate-activated openings exhibited conductances of 3–15 pS, falling within the range activated by glutamate and AMPA in these cells. In contrast, while SYM 2081 (10, 50 and 100 µM) also activated a high frequency of brief events, it gave a greater proportion of well-resolved higher-conductance openings (>15 pS) (Figs 8C and 11A).
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= 6.1 pS in 30 µM kainate, compared with
= 10.9 pS in 100 µM SYM. The mean channel conductance for all patches examined was
= 6.7 ± 0.8 pS (n
= 4) in 30 µM kainate, compared with
= 11.8 ± 0.6 pS in 100 µM SYM (n
= 7).
In marked contrast with the other agonists examined, the shut time histograms for all patches exposed to kainate were well fitted with a single exponential with
= 4.6 ± 1.3 ms (n
= 4) (see Fig. 9A). On the other hand, for SYM 2081-activated events (Fig. 9C), the shut time histograms were best fitted with three exponential components, with 1
= 0.13 ± 0.02 ms (7 ± 2%), 2
= 9.5 ± 1.3 ms (57 ± 9%) and 3
= 133 ± 11 ms (37 ± 7%) (n
= 7).
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1
= 0.28 ± 0.05 ms (49 ± 1%) and 2
= 1.1 ± 0.1 ms (51 ± 3%) (n
= 4); and in 100 µM SYM 2081, 1
= 0.23 ± 0.01 ms (73 ± 2%) and 2
= 1.2 ± 0.07 ms (27 ± 2%) (n
= 7). The absence of a short shut time component in patches exposed to kainate reflected the absence of resolvable short closings within channel activations, so that channel openings did not display bursts. Although a short shut time component could be detected in channels activated by SYM 2081 (see Fig. 9C), its area (7 ± 2%) was relatively small. We did not therefore attempt to estimate a burst distribution for these patches.
Compared with channels activated by AMPA or glutamate, there was a clear dominance of brief openings and a lack of bursts in the presence of kainate and SYM 2081. Furthermore, the open periods of channels activated by kainate and SYM 2081 displayed some marked similarities, with fewer of the long open periods that characterized AMPA- and glutamate- (10 µM or 20 mM) activated events.
These observations, in particular the activation of brief openings in response to low concentrations of SYM 2018 (10 µM; Fig. 10A), further supported the view that brief events arose mainly from kainate receptors, while the concentration-dependent ‘bursty’ events arose from AMPARs in CA1 cells. To test this hypothesis, we next examined channels activated by SYM 2018 (Fig. 10B) and by glutamate (Fig. 11) in the presence of the highly selective AMPAR antagonist GYKI 53655.
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As is apparent in Fig. 10B, following application of GYKI 53655 (100 µM) channels are still activated by 30 µM SYM 2018, consistent with the view that these brief events arose mainly from kainate receptors. However, the application of GYKI 53655 (Fig. 11A) led to a marked decrease in the macroscopic current stimulated by a steady application of 20 mM glutamate. Figure 11B shows examples of the underlying single-channel currents on a faster time base. The reduction in channel activity was characterized by a marked decrease in the long-duration (‘bursty’) openings in the presence of GYKI 53655.
To quantify this observation, we applied a detailed analysis to three patches exposed to glutamate (10 mM) in the absence and presence of GYKI 53655. The properties of the channel openings were dramatically changed in the presence of GYKI. This is illustrated in Fig. 11C, which shows a typical open period distribution in a patch before (shaded histogram) and after (open histogram) GYKI 53655 application. For all patches examined, the majority of glutamate-activated channels measured in the presence of GYKI displayed a reduced contribution from longer-duration events, and an increased proportion of short open periods.
The open period histogram was best fitted with the sum of two exponentials. The mean time constants were, 1
= 0.19 ± 0.18 ms (94 ± 3%) and 2
= 2.1 ± 0.6 (6 ± 1%) (n
= 3). Surprisingly, the number of transitions during a given period was similar in steady glutamate application, and in the presence of glutamate with GYKI 53655. Furthermore, GYKI 53655 reduced the mean channel conductance from
= 11.2 ± 1.5 pS (n
= 6) in the presence of glutamate (Vm
=
–100 mV), to
= 6.9 ± 2.0 pS (n
= 3) in glutamate with GYKI. The latter value closely approximates the channel conductance activated by 30 µM kainate (6.7 pS). These observations are consistent with the view that the ‘bursty’ events arose predominantly from AMPARs in CA1 neurones, and that brief openings arose from kainate receptors.
Multiple-conductance events were more prevalent at higher agonist concentrations
During an individual event, many channel openings adopted several different conductance levels before finally closing. When there is more than one channel in the patch it can be difficult to differentiate the presence of multiple-conductance levels from superimposed openings (Traynelis & Jaramillo, 1998). However, it was of interest to try to obtain an idea of the number of possible conductance levels that could be adopted during an individual opening. Furthermore, it was also of interest to determine whether the number of events displaying multiple conductances increased with higher glutamate concentrations. Figure 12A illustrates examples of individual channel openings (activated by 20 mM glutamate), that have been selected to show the presence of two, three or four different conductance levels. To determine the proportion of events displaying multiple conductance levels we estimated the ratio of the number of openings to the number of open periods (see Fig. 12B for details).
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To exclude the possibility that an increased prevalence of superimposed openings could account for the observed correlation between increased glutamate (or AMPA) concentration, and the number of openings/number of open periods (Fig. 12C), we estimated the probability that openings in a patch were superimposed (see Appendix). Using this statistical approach we demonstrated that the correlation between agonist concentration and the mean ratio of the number of conductance levels adopted during an individual opening occurred independently of the increased probability of superimposed openings.
Analysis of conductance levels and direct transitions
Analysing the amplitudes of the single-channel currents associated with AMPA and kainate receptors was hampered by the following problems: (1) the conductances were small and openings were brief, limiting our resolution; (2) a variety of different conductances were present in each patch; and (3) there was some patch-to-patch variation. Nevertheless, from the fitted Gaussians we could identify 3–6 (out of 7) different conductance levels in all of the patches exposed to 10 µM or 10 mM glutamate, and 1 µM or 10 µM AMPA.
The conductance values (mean ± S.D.) for each concentration are plotted in Fig. 13A. These values were obtained by identifying a particular conductance level from its fitted Gaussian. The mean for this Gaussian was then averaged across patches. We found no significant difference in the identified conductance values for different concentrations of AMPA and glutamate (Student's t test, P > 0.05).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionAppendixReferences |
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Distinct AMPA- and kainate-type receptor channels are present in CA1 cells
Channel properties of a wide variety of AMPA and kainate receptor subtypes have previously been described in recombinant systems (Howe, 1996; Swanson et al. 1996, 1997; Derkach et al. 1999; Banke et al. 2000; Oh & Derkach, 2005), and in identified neurones in slices (Jonas & Sakmann, 1992; Spruston et al. 1995; Banke et al. 2000; Smith & Howe, 2000; Momiyama et al. 2003). For single-channel analysis, we distinguished between kainate and AMPAR channels using the selective kainate receptor agonist SYM 2081, which activated brief openings that did not occur in bursts. These events could also be activated by kainate and glutamate, and displayed properties consistent with the view that they arose from kainate receptors: (1) their fast kinetics resembled those of recombinant kainate receptor channels (Swanson et al. 1996); (2) they appeared to lack concentration-dependent conductance, in agreement with data on kainate channels in cerebellar granule cells (Smith & Howe, 2000); (3) they were resistant to block by GYKI 53655 (AMPAR antagonist). In contrast, AMPARs gave rise to distinct ‘bursty’ openings that could be selectively suppressed by GYKI 53655. These events were noticeable in the presence of glutamate, consistent with previous observations suggesting that glutamate activates a preponderance of AMPARs in CA1 cells (Spruston et al. 1995).
AMPAR bursts of openings display concentration-dependent conductance and kinetics
We have shown that the channel conductance activated by AMPA and glutamate increases with agonist concentration. This finding is in keeping with previous work on a recombinant chimeric AMPAR–kainate receptor (Rosenmund et al. 1998) and on native AMPARs in developing cerebellar granule cells (Smith & Howe, 2000). In patches from CA1 pyramidal cells, we obtained a mean conductance of
= 7.7 pS when channels were activated by 10 µM AMPA. This matches well the value (7.9 pS) obtained previously using analysis of stationary noise induced by 30 µM AMPA in patches (Jonas & Sakmann, 1992).
During transmission in the hippocampus and cerebellar cortex, the glutamate transient within the cleft is thought to follow a biphasic decay (Silver et al. 1996; Diamond & Jahr, 1997, 2000; DiGregorio et al. 2002), attaining an average peak concentration of roughly 3 mM. This is suggested to decay rapidly (
= 100 µs) to a more slowly decaying component of 0.5 mM (Bergles et al. 1999; Mainen et al. 1999). Interestingly non-stationary noise analysis of responses to fast application of 1 mM glutamate in CA1 dendritic patches, gave a mean AMPAR channel conductance of
= 10.2 pS (Spruston et al. 1995). This estimate exceeds slightly the value obtained by Jonas & Sakmann (1992), in keeping with our present observations that channel conductance of AMPARs in CA1 cells is concentration dependent. Furthermore, this estimate matches well our mean value obtained from single-channel analysis. It is of note that we observed roughly a doubling of mean channel conductance from 5 pS in 200 nM glutamate to 11 pS in 10 mM glutamate.
We cannot be sure that the extrasynaptic channels that we have examined in the somatic membrane of CA1 cells represent a homogeneous population, or that their properties will be identical to those of the synaptic receptors. Furthermore, it could be argued that the higher-conductance events we observed somehow arose from the presence of a separate population of channels activated only at high glutamate concentration. This seems unlikely for several reasons. First, such a channel would have an unusually high EC50. Second, the change in Gaussian distribution that we observe does not fit well with this idea: the accompanying reduction in the lower-conductance openings would be unexpected unless they desensitize. Third, and most compelling, we see transitions between the low- and high-conductance levels in all patches examined. This implies such events arise from the same receptor channel.
Kainate receptors do not give rise to bursts of openings
Our experiments strongly suggest that the kainate receptors in CA1 cells are composed of GluR6 and KA2 subunits as previously suggested (Wisden & Seeburg, 1993a). Thus, our estimated kainate channel conductance,
= 6.7 pS, matches well to our previous estimate of
= 7.1 pS obtained from recombinant kainate receptor assemblies containing GluR6/KA2 subunits (Swanson et al. 1996). Our conductance estimate for kainate receptor channels activated by 30 µM kainate, exceed the value of 3.6 pS obtained using analysis of fluctuations induced by 300 µM kainate (Jonas & Sakmann, 1992). The fast kinetics that we observed for kainate-activated openings, may well cause attenuation of conductance estimates obtained from noise analysis. Although the resolution of both methods is similar, events that were too brief to reach full amplitude could be readily identified, and therefore excluded from the single-channel analysis.
The open periods obtained with kainate or SYM 2081 in CA1 cells displayed time constants that were in the same range as described for recombinant receptors containing GluR6/KA2 subunits (Swanson et al. 1996). The higher conductance value obtained for events activated by 100 µM SYM 2081 (
= 11.8 pS), might suggest that this concentration of SYM 2081 activates both kainate and AMPARs (Donevan et al. 1998) in these cells, to give a conductance estimate weighted towards the higher levels.
The AMPAR antagonist GYKI 53655 reduced the amplitude and mean open time of glutamate-activated channels, while leaving their frequency of opening virtually unchanged. This supports the view that ‘bursty’ events arose from AMPARs. However, the fact that the frequency was unaltered may reveal information about the action of GYKI 53655. Three possible explanations could account for our observations: (1) the remaining channel openings may arise from kainate receptors; (2) GYKI 53655 may cause ‘flickery block’ of AMPAR channels; or (3) brief low-conductance activations may be counted more efficiently during relatively low channel activity in the presence of GYKI 53655.
Multiple-conductance events at higher agonist concentrations
Single-channel recordings presented in this paper have shown that during an individual channel opening, up to four different conductance states can occur. Previously it has been proposed that the current mediated by the opening of a single AMPA receptor channel is correlated to the number of glutamate molecules bound to the receptor. With two molecules bound, the channel opened to a small conductance; with three molecules bound, a medium conductance was activated; and with four bound molecules (fully liganded) a high conductance was activated (
