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Institute of Basal Medicine, Department of Physiology, and Centre of Molecular Biology and Neuroscience, University of Oslo, Norway
Submitted 2 February 2008; accepted in final form 31 July 2008
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ABSTRACT |
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INTRODUCTION |
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). These channels are composed of proteins of the KCa2 family, the three members (KCa2.1–2.3, also called SK1-3) of which are all expressed in the mammalian brain, including the hippocampus (Kohler et al. 1996; Sailer et al. 2002; Stocker and Pedarzani 2000). The selective SK channel blocker, apamin, a bee venom peptide toxin, has been reported to improve learning and memory retention of mice, facilitate the induction of hippocampal long-term synaptic potentiation (LTP), and increase immediate early gene expression (Behnisch and Reymann 1998; Heurteaux et al. 1993; Kramar et al. 2004; Messier et al. 1991; Norris et al. 1998; Stackman et al. 2002). Dysfunction of SK channels may be linked to psychiatric disease, including schizophrenia and bipolar disorder (Chandy et al. 1998), and these channels may also be targets of antipsychotic drugs (Terstappen et al. 2001). It is therefore of considerable interest to determine the physiological roles at the cellular level of neuronal SK channels in the brain.
The main functions usually ascribed to SK channels in neurons, are generation of afterhyperpolarizations (AHPs) and feed-back regulation of neuronal excitability, including spike-frequency adaptation (Bond et al. 1999; Hille 2001; Sah 1996; Sah and Faber 2002; Vergara et al. 1998). Rodent hippocampal pyramidal cells have been widely used as prototype neurons for studying mechanisms and functions of AHPs in general and SK channels in particular (for review, see e.g., Bond et al. 2005; Faber and Sah 2003, 2007; Storm 1990). In these cells, action potentials are followed by a sequence of three AHPs: fast (fAHP), medium (mAHP), and slow (sAHP) (Storm 1987a, 1989). A similar pattern is found in many other mammalian central neurons (Pape and Driesang 1998; Pineda et al. 1992; Schwindt et al. 1988; Takahashi 1990; Viana et al. 1993).
Until a few years ago, the hippocampal sAHP, which is modulated by a wide variety of transmitter and second-messenger systems, was generally believed to be caused by SK channels (Hille 2001; Kohler et al. 1996; Vergara et al. 1998). In particular, channels formed by SK1 subunits, which initially were reported to be rather insensitive to apamin (Kohler et al. 1996), were considered likely generators of the apamin-resistant sAHP (Hille 2001; Vergara et al. 1998). However, recent findings indicate that SK channels do not underlie the sAHP after all (for review, see Vogalis et al. 2003): SK1 channels were found to be far more sensitive to apamin than the sAHP (Shah and Haylett 2000; Strobaek et al. 2000), overexpression of SK1 or SK2 in neocortical pyramidal cells did not enhance IsAHP and expression of a dominant negative SK3 subunit, capable of suppressing SK1, SK2, and SK3 channels, did not reduce IsAHP (Villalobos et al. 2004), and CA1 pyramidal cells of transgenic mice lacking functional SK1, SK2 and SK3 genes, still showed robust sAHPs (Bond et al. 2004).
In view of the highly probable conclusion that the SK channels do not generate the sAHP and that these channels nevertheless can generate a considerable K+ current in hippocampal pyramidal cells (Bond et al. 2004; Sah and Faber 2002; Sailer et al. 2002; Stackman et al. 2002; Stocker et al. 1999; Vogalis et al. 2003), it is important to determine the functions of this channel type.
Almost 20 years ago, the SK channel blocker apamin was first used to test the hypothesis that SK channels generate somatic AHPs and excitability control in hippocampal pyramidal cells, but the results were consistently negative (Storm 1989; Williamson and Alger 1990). Instead these studies indicated that the mAHP in these cells was generated by M channels when the cell is depolarized and by h channels when it is hyperpolarized (Storm 1989). This conclusion was supported by a recent study of transgenic mice, where the mAHP of CA1 pyramidal cells was strongly reduced when M/KCNQ channels were suppressed by expression of dominant negative KCNQ2 subunits (Peters et al. 2005). Furthermore Gu et al. (2005) confirmed and extended the conclusions from (Storm 1989) that the somatic mAHP in rat CA1 pyramidal cells is not caused by SK channels but is mainly generated by Kv7/KCNQ/M channels at depolarized levels and by HCN/h channels at hyperpolarized levels. Also Yue and Yaari (2004), studying the roles of potassium currents in control of excitability, afterdepolarizations, and bursting in rat CA1 pyramidal cells, found clear effects of Kv7/KCNQ/M channel blockers but no effect of SK channel blockers.
In spite of these convergent lines of evidence, there is still a persistent and widespread view that SK channels contribute substantially to the mAHP in rat CA1 hippocampal pyramidal neurons (Bildl et al. 2004; Bond et al. 1999, 2004; Bowden et al. 2001; Faber and Sah 2003; Melyan et al. 2002; Sah and Faber 2002; Sailer et al. 2002; Stocker 2004; Stocker et al. 1999, 2004). Thus until very recently, several studies have claimed that an apamin-sensitive current contribute to the mAHP and excitability control in these neurons (Bond et al. 2005; Faber and Sah 2007; Kaczorowski et al. 2007; Shah et al. 2006). Resolving this issue seems essential for understanding the functions of this prominent channel family in an important and widely studied class of neurons. Therefore we have now re-examined the functions of SK channels in hippocampal pyramidal cells under a variety of experimental conditions. Consistently we found no detectable contribution to the mAHP or excitability control from SK channels under any of the conditions tested.
Next we asked the question if the SK channels in CA1 pyramidal cells are not generating the sAHP or the mAHP or excitability control or spike frequency adaptation, as accumulating evidence now indicates (Gu et al. 2005; Shah and Haylett 2000; Storm 1989; Strobaek et al. 2000; Villalobos et al. 2004; Vogalis et al. 2003), what else is their function in these cells?
Interestingly, recent studies of pyramidal cells from both hippocampus and amygdala indicate that dendritic SK channels are activated by stimulation of glutamate receptors in the dendrites by glutamatergic synaptic input or artificially applied glutamate; these SK channels are activated presumably via calcium influx through NMDA receptors and/or voltage-gated calcium channels; and the SK current can limit calcium influx by repolarizing the cell and reinstate the Mg2+ block of the N-methyl-D-aspartate receptor (NMDAR) channels, thus forming a negative feedback loop (Bloodgood and Sabatini 2007; Stackman et al. 2002). These and other data indicate that SK channels may regulate and even contribute to hippocampal synaptic plasticity (Faber et al. 2005; Lin et al. 2008; Ngo-Anh et al. 2005; Stackman et al. 2002; Tzounopoulos and Stackman 2003). Our data support such postsynaptic functions of dendritic SK channels.
We conclude that postsynaptic SK channels in rat CA1 pyramidal cells can be activated by dendritic calcium influx through NMDA receptors and/or voltage-gated Ca2+ channels but cannot be sufficiently activated by normal somatic sodium action potentials to affect somatic responses in this cell type under a variety of conditions. Our data seem to indicate that in this cell type somatic depolarization can substantially activate SK channels only under artificial or extreme conditions, such as following Ca2+ spikes facilitated by potassium channel blockers or in the presence of an SK channel opener drug.
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METHODS |
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Transverse hippocampal slices (400 µm thick) were prepared from male Wistar rats (2.5–8 wk of age). Also older Wistar rats (
6 mo) were used in some experiments (Table 1). The experimental procedures were approved by the responsible veterinarian of the Institute, in accordance with the statute regulating animal experimentation (Norwegian Ministry of Agriculture 1996). Briefly, the rats were deeply anesthetized with Suprane before decapitation. Hippocampal slices (400 µm) were cut with a vibratome (Campden Instruments, UK) and maintained in an interface chamber filled with artificial cerebral spinal fluid (ACSF) containing (mM) 125 NaCl, 25 NaHCO3, 1.25 KCl, 1.25 KH2PO4, 1.5 MgCl2, 1.0 CaCl2, and 16 glucose and saturated with 95% O2-5% CO2. During recording, the slices were kept submerged and perfused with ACSF of the composition described in the preceding text, except that [CaCl2] was 2.0 mM. To block Ca2+ influx, the ACSF was sometimes replaced by Ca2+-free medium containing (in mM) 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.5 MgCl2, 2.0 MnCl2, and 16 glucose. In some experiments, 10 µM 6,7-dinitroquinoxaline-2,3-dione (DNQX) was added to the ACSF to block spontaneous excitatory synaptic transmission. The ACSF was saturated with 95% O2-5% CO2, and the temperature was kept constant (within ±0.5°C), normally at 30°C.
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, pH 7.3–7.4). Whole cell recordings were obtained under IR-DIC microscopy or with the "blind" method. The patch pipettes were filled with a solution containing (in mM) 140 KMeSO4 or K gluconate, 10 HEPES, 10 phosphocreatine Na salt, 2 ATP Na salt, 0.4 GTP Na salt, and 2 MgCl2 (pipette resistance, 4–7 M; series-resistance, Rseries, 10–40 M). All potentials were corrected for the junction potential. For perforated-patch recording, the pipette solution contained (in mM) 140 KMeSO4, 10 HEPES, 2 ATP, 0.4 GTP, 2 MgCl2, 10 phosphocreatine, and 10 tetraethylammonium chloride (TEA) and 50 µg/ml gramicidin. After seal formation (>1 G seal resistance), we saw a gradual reduction in Rseries, to <100 M. The recording was discarded if we observed a precipitous drop in Rseries, considered to indicate a break-in to whole cell configuration. By using TEA in the pipette and monitoring spike half-width, we obtained an additional assessment of subtle perforated patch rupture. Only cells with a stable resting membrane potential negative to –60 mV and stable action potential duration and amplitude (>80 mV) were used for recording.
To evoke excitatory synaptic potentials in the CA1 pyramidal cells in whole cell mode, presynaptic fibers were electrically stimulated with a sharpened tungsten electrode placed in the middle of stratum radiatum 
100 µm from the cell recorded (stimulus: 100 µs, 70–250 µA). Trains of five stimuli at 100 Hz were delivered once every 30 s. Field potentials were recorded by stimulation of presynaptic fibers with a tungsten electrode and recording in the middle of the st. radiatum with a low-resistance pipette. During whole cell recordings and field potential recordings in Figs. 3 and S4,1 DL-2-amino-5-phosphonopentanoic acid (DL-AP5, 100 µM) was routinely added to the ACSF to prevent long-term potentiation (LTP) or depression (LTD). In the experiments illustrated in Fig. 7, the extracellular Mg2+ concentration was reduced to 1.0 mM, and DNQX (40 µM) and picrotoxin (100 µM) were added to the bath solution to block the non-NMDAR-mediated excitatory postsynaptic potentials (EPSPs) and GABAR-mediated inhibitory postsynaptic potentials (IPSPs), to isolate NMDAR-mediated EPSPs. Short trains of stimuli (3 stimuli at 100 Hz) were delivered once every 20 s. D-AP5 (100 µM) was added before the end of every experiment (n = 12) to confirm that the EPSPs were mediated by NMDA type glutamate receptors.
Data acquisition, storage, and analysis
The data were acquired with pCLAMP 7.0 or 9.0 (Molecular Devices) at a sampling rate of 5–20 kHz and were measured and plotted with pCLAMP 9.0 and Origin 7.0 (Microcal). Values are expressed as means ± SE. Two-tailed Student's t-test was used for statistical analysis (
= 0.05). The P values are given in the figure legends.
The mAHP and sAHP amplitudes were measured by averaging the values within a time window (20 and 50 ms, respectively) around the peak of each AHP (20–100 ms and 0.1–3.0 s after a spike or spike train, respectively). The cell input resistance was measured by injecting weak negative current pulses and dividing the steady-state voltage response by the current pulse amplitude (Rinput = 
V/I).
Chemicals and drugs
Apamin was obtained from Latoxan (France), XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone] from DuPont, DNQX and 1-ethyl-2-benzimidazolinone (1-EBIO) from Tocris Cookson, and the remaining chemicals from Sigma-Aldrich Norway AS (Oslo, Norway). Substances were bath-applied by adding them to the perfusion medium.
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RESULTS |
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The resting membrane potential (Vrest) and input resistance (Rinput) were –64.5 ± 0.7 mV and 44.6 ± 1.8 M for sharp electrode recordings (n = 30), –72.8 ± 1.4 mV and 50.8 ± 2.6 M for whole cell recording (n = 10), and –74.3 ± 1.2 mV and 79.1 ± 4.1 M for perforated patch recordings (n = 5), respectively.
SK channels are selectively activated by Ca2+ action potentials but not by Na+ spikes
To ensure that the SK channels were available for activation under our experimental conditions and that our apamin or application procedure was effective, we first used experimental paradigms that are known to strongly activate Ca2+-activated K+ channels (Fig. 1A).
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, 1986; Pedarzani and Storm 1993). Bath application of 100 nM apamin fully blocked the medium AHP (
, mAHP), whereas the slow AHP was resistant to apamin (
, sAHP) in agreement with previous reports (Gu et al. 2005; Lancaster and Nicoll 1987; Stocker et al. 1999; ; Storm 1989). Apamin also strongly increased the duration of the Ca2+ spike and caused a prominent afterdepolarization (ADP, *) with additional Ca2+ spikes to appear, indicating blockade of an SK current that contributed to repolarization (Fig. 1A). Similar effects of apamin were found in all cells that showed a prominent mAHP (>2.0 mV) following the Ca2+ spike (n = 5; see summary data in Fig. 1C). In some cells, the mAHPs following Ca2+ spikes were small (<2.0 mV) or absent, apparently being partly occluded or replaced by an afterdepolarization (ADP), which may be due to slow decay of the inward Ca2+ current, and/or activation of the Ca2+-activated nonselective cation current (ICAN); (Knox et al. 1996). Nevertheless apamin showed clear effects also in these cells, by suppressing the mAHP and/or enhancing the ADP. Supplementary Fig. S1 shows the average time course of apamin effect in all cells tested (including cells with and without a prominent mAHP: n = 10). In contrast, when cells were exposed to normal extracellular ACSF (without TTX and TEA), apamin failed to affect the mAHPs that followed 50-ms high-frequency Na+ spike trains (Fig. 1, B and D; n = 6, P > 0.05).
The effect of apamin on Ca2+ spike-mAHPs was seen also when our recording prior to apamin lasted longer (10–12 min; n = 5) than in the sharp electrode recordings without TTX or TEA where apamin failed to affect the mAHP (8–10 min). This, as well as our previous recordings of robust apamin-sensitive tail currents under similar conditions (Sailer et al. 2002), indicates that channel rundown was not the cause of the negative results. Evidently, SK channels are available for activation in our recording conditions, but they require exceptionally strong Ca2+ influx to noticeably affect somatic electrical activity.
Taken together, our results indicate that functional SK channels are available in CA1 pyramidal cells and can be activated by the massive Ca2+ influx provided by Ca2+ spikes evoked in the presence of Na+ and K+ channel blockers, but that these SK channels are not significantly activated by Na+ spike trains under normal conditions at least not to an extent that appreciably contributes to the somatic mAHP.
Do SK channels contribute to the mAHP under other experimental conditions?
Although several studies have indicated that the somatic mAHPs in CA1 pyramidal neurons are not caused by SK channels, but mainly by M current at depolarized potentials and h current at hyperpolarized potentials (Gu et al. 2005; Peters et al. 2005; Storm 1989), apparently conflicting results have been reported and widely cited by others (Bildl et al. 2004; Bond et al. 1999, 2004, 2005; Bowden et al. 2001; Faber and Sah 2003, 2007; Kaczorowski et al. 2007; Melyan et al. 2002; Sah and Faber 2002; Shah et al. 2006; Stocker 2004; Stocker et al. 2004). Therefore we found it necessary to test the roles of SK channels systematically under a variety of experimental conditions. Hence we conducted an extensive search for an SK-channel contribution to the mAHP (n = 77 cells; Table 1), while varying parameters that might have caused different results: age of the animal, temperature, recording technique (whole cell gigaseal, sharp electrode intracellular, and perforated-patch recording), extracellular [K+], [Ca2+], and pH, background membrane potential, duration of the depolarizing pulse, number and frequency of action potentials, and used different SK channel blockers. All tests were performed in the absence of bicuculline because some bicuculline salts are known to suppress SK channels (Johnson and Seutin 1997).
The results of these tests are summarized in Table 1. Figure 2 shows typical recordings and averaged time courses obtained with sharp electrodes in slices from 3- to 8-wk-old rats, in the presence of 2 mM [Ca2+]o. Application of apamin produced no detectable effect on the mAHP and sAHP in any of the conditions tested. For example, apamin failed to affect the mAHP and sAHP following a high-frequency train of five spikes evoked by a depolarizing current pulse (50 ms) in 3.5 mM [K+]o at 30°C with the background membrane potential (Vm) held at –60 mV (Fig. 2A). A high concentration of the SK channel blocker d-tubocurarine (d-TC, 100 µM) had no detectable effect on the mAHP or sAHP following a longer spike train (400 ms) at room temperature in 2.5 mM [K+]o (Fig. 2B). This protocol resembles closely to the one used by Stocker et al. (1999), e.g., their Fig. 5. Apamin had no significant effect on the mAHP following a high-frequency (100 Hz) train of 20 spikes evoked by 20 brief (2 ms) pulses in 2.5 mM [K+]o at 30°C (Fig. 2C). Apamin also failed to affect the mAHP following five spikes in perforated-patch recordings in 2.5 mM [K+]o at 30°C (Fig. 2D). Finally, apamin had no effect on the mAHP following a single spike in 2.5 mM K+ at 26°C (Fig. 2E). In contrast, the M-channel blocker XE991 consistently abolished the mAHP at this membrane potential (Gu et al. 2005; Peters et al. 2005) (Fig. 2E).
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), apamin failed to affect the mAHP (n = 7, Table 1, pHo). However, in some experiments, we observed a slow, time-dependent rundown of the mAHP amplitude, independently of drug application, in particular during whole cell recording. A typical example of such rundown (from a cell that was excluded from analysis for this reason) is illustrated in Supplementary Fig. S2. Such rundown may perhaps explain why others have reported an apparent reduction in mAHP following apamin application during whole cell recording.
Taken together, these results support the conclusion that SK channels do not contribute noticeably to the mAHP in rat CA1 pyramidal cells.
Ionic mechanisms of mAHPs following excitatory synaptic activation
Although SK channels do not seem to contribute appreciably to the normal mAHP evoked by somatic current injection, it is still possible that excitatory synaptic input could activate these channels. This seems an interesting possibility because the apical dendrites that receive most of the excitatory synaptic input are also prone to generate Ca2+ spikes or Ca2+ channel-mediated plateau potentials (Andreasen and Nedergaard 1996; Cai et al. 2004; Magee and Carruth 1999; Magee and Johnston 1995).
To test this hypothesis, presynaptic axons in st. radiatum were activated by high-frequency trains of stimuli, each evoking an EPSP. First, we tested whether SK channels are activated by EPSPs caused by non-NMDA-type glutamate receptors by stimulating in the presence of the NMDA receptor blocker APV (Fig. 3A, control) . To maximize the postsynaptic Ca2+ influx and, thus the likelihood that SK channels should be activated, the stimulation intensity was increased until each EPSP evoked an action potential. The train of five EPSPs with spikes was followed by a mAHP; but application of 100 nM apamin had no detectable effect (Fig. 3, A, apamin, and B, left). In contrast, XE991 readily blocked the synaptically evoked mAHP, and uncovered an afterdepolarization (ADP *; Fig. 3, A, B, right, and C). Similar negative results with apamin were obtained in all five cells tested (see the summary graphs in Fig. 3, D and E).
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To avoid possible side effects of intracellular recording, including possible rundown of Ca2+ channel or SK channel activity, we also performed extracellular field potential recordings from st. pyramidale in CA1 (Supplementary Fig. S4). Stimulation of axons in st. radiatum with high-frequency (100 Hz) trains of six pulses at high stimulus intensity evoked large field EPSPs and population spikes. Bath application of 100 nM apamin had no detectable effect on these field potential responses (Supplementary Fig. S4). Taken together, these results indicate that not even the spikes and subsequent mAHPs triggered by excitatory dendritic synapses are appreciably modulated by SK channels when the NMDA-receptors are blocked (see following text).
Excitability control and spike frequency adaptation
To further test how K+ currents underlying the mAHP affect excitability, we evoked repetitive firing by injecting 1-s depolarizing pulses. As illustrated in Fig. 4A, top, apamin (100 nM) had no significant effect on the number, frequency, or pattern of action potentials, whereas XE991 (10 µM) strongly increased the spike frequency, often causing an initial high-frequency spike cluster, followed by a pause in the firing and an enhanced sAHP (Fig. 4A, right). Similar results were obtained for different current intensities and spike frequencies (Fig. 4A, middle and bottom). As illustrated in Fig. 4B, when the spike frequency was plotted as a function of injected current intensity (f/I plots) for all the five cells tested, no obvious effect of apamin could be detected, whereas XE991 clearly increased the slopes of the f/I curves, indicating a significant increased excitability (Fig. 4B, right). This was seen both for the instantaneous frequency of the three first interspike intervals (1/ISI) as well as for steady-state discharge frequency toward the end of each pulse (Fig. 4B). Likewise, when the average f/I plots were compared before and after apamin and XE991 applications (Fig. 4D), no significant effect of apamin could be detected, whereas XE991 increased the action potential (AP) frequency at all current intensities tested (Fig. 4D, right). Finally, when the instantaneous discharge frequency was plotted as a function of time during the spike train (Fig. 4C, current pulses: 0.15 nA), no effect of apamin was apparent, whereas XE991 clearly increased the firing frequency (Fig. 4C, right).
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These results indicate that SK channels do not contribute appreciably to excitability control or spike frequency adaptation in CA1 pyramidal cells.
Effects of the SK-channel opener 1-EBIO
To further test whether SK channels in CA1 pyramidal cells were available for activation under our experimental conditions and how they might contribute if they were activated, we used the SK-channel opener 1-EBIO (Pedarzani et al. 2001). As shown in Fig. 5, application of 10 µM 1-EBIO more than doubled the amplitude of the mAHP following a five-spike train (Fig. 5, A–C). Subsequent application of apamin, in the continued presence of 1-EBIO (Fig. 5, A–C), exactly reversed the effect of 1-EBIO, thus returning the mAHP to the same amplitude as it has in control medium (Fig. 5, A–C). In parallel, 1-EBIO, reduced the excitability of the cell, so that a stronger current pulse was needed to evoke five spikes in the presence of 1-EBIO than in control medium or after apamin had been added (Fig. 5A, bottom). By comparing the response to the same current pulse intensity before and after adding 1-EBIO, the 1-EBIO-induced reduction in excitability was clearly seen as a reduction in the number of spikes (Fig. 5E). This effect was also reversed by adding apamin (Fig. 5F). However, no resting membrane potential (RMP) change was detected after application of 1-EBIO, and the input resistance (calculated by dividing the steady-state voltage response by the current pulse amplitude: Rinput = V/I) was also the same before (55.5 ± 6.25 M) and after (55.6 ± 5.9 M) application of 1-EBIO (paired t-test, P > 0.05, n = 4). The latter result is probably due to lack of SK channel activation at resting intracellular [Ca2+] levels, even in the presence of 1-EBIO.
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Because our present and previous data (Gu et al. 2005; Storm 1989) indicate that SK channels do not contribute appreciably to the mAHP following Na+ spikes in CA1 pyramidal cells, we wished to test whether this is generally true also for other classes of hippocampal pyramidal cells. In a parallel study, we have tested CA3 pyramidal cells and found that apamin has no obvious effect on the Na+-spike-induced mAHP also in these cells (Sharifullina et al. 2008). In addition, we tested pyramidal neurons in the subiculum area of the rat hippocampus (Fig. 6). Most subicular pyramidal cells show bursting behavior, firing clusters of action potentials at high frequency (>200 Hz) (Staff et al. 2000). Only such bursting cells were included in this study. Figure 6A shows the typical firing patterns of such a bursting subicular neuron. We found that bath application of apamin (100 nM) significantly reduced the mAHP following the burst in these subicular pyramidal neurons (Fig. 6B). Thus an apamin effect was observed in all cells tested (Fig. 6, C and D, n = 8, P < 0.01), in clear contrast to the lack of effect in CA1 pyramidal cells. (A preliminary report of these results was published by Hu et al. 2004.) These results indicate that the functional roles of SK channels differ between bursting subicular and other hippocampal (CA1, CA3) pyramidal cells. In addition, these results indicate that there is nothing about our recording methods, conditions or slice preparations that prevent us from observing an apamin effect on the mAHP when it occurs. Thus the CA1 cells and subicular cells were recorded in identical horizontal hippocampal slice preparations under identical conditions. In a previous study, parallel recordings of apamin effects on AHPs in frog sympathetic neurons served as a similar positive control (Storm 1989).
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Because the SK channels in CA1 pyramidal cells appear not to generate the mAHP or the sAHP (Gu et al. 2005; Shah and Haylett 2000; Storm 1989; Strobaek et al. 2000; Villalobos et al. 2004; Vogalis et al. 2003), we next asked what else their function might be. Previous studies have provided evidence that dendritic SK channels activated by Ca2+ influx through NMDA receptors or voltage-gated Ca2+ channels can regulate dendritic Ca2+ signals and modulate synaptic efficacy and plasticity (Bloodgood and Sabatini 2007; Faber et al. 2005; Ngo-Anh et al. 2005; Stackman et al. 2002). In particular, Ngo-Anh et al. (2005) found that apamin increased the peak EPSP amplitude in mouse CA1 pyramidal cells by 70% (in the presence of 1 mM Mg and picrotoxin), and subsequent application of APV reversed the apamin-induced increase. Thus it appears that SK channels, by generating a repolarizing conductance in response to increased intracellular Ca2+, form an effective Ca2+-mediated feedback loop that regulate postsynaptic Ca2+ influx. These conclusions are based on results from hippocampal slices from mice (Bloodgood and Sabatini 2007; Stackman et al. 2002), rat hippocampal slice cultures (Cai et al. 2004), acute slices from rat hippocampus (Ngo-Anh et al. 2005) and rat amygdala (Faber et al. 2005).
To test, in a noninvasive manner, whether SK channels are activated by NMDAR-mediated synaptic transmission and can regulate its impact, we performed extracellular field potential recordings in st. radiatum of CA1. Figure 7A shows NMDAR-mediated postsynaptic responses (EPSPs) in normal medium with 1 mM [Mg2+] evoked by a stimulation electrode in st. radiatum, which contains the Shaffer collateral pathway from CA3 to CA1. The responses were induced by a series of three stimuli at 100 Hz in the presence of the AMPA receptor antagonist DNQX (40 µM) and the GABAA receptors blocker picrotoxin (100 µM). Subsequent application of the SK channel blocker apamin (100 nM) significantly increased the amplitude and area of the EPSPs (red trace). Finally, application of the NMDA receptor antagonist DL-AP5 blocked the EPSPs (blue trace). The average time courses of the apamin effects on the peak amplitude, the area, and the decay time of the last NMDAR-EPSPs are shown in Fig. 7, B–D, respectively (n = 6, P < 0.05). Note, however, that the apparent temporal summation of NMDAR-mediated EPSPs is highly nonlinear, both before and after apamin application (see Fig. 7A, inset). Thus the EPSP component evoked by the third stimulus is far larger than the two previous ones, and it also shows a remarkably slow rise and a "noisy" peak. Although the increased amplitude of the late EPSPs may be partly due to presynaptic facilitation, the effect is so large that it seems likely to involve activation of voltage-dependent dendritic conductances, such as regenerative depolarizations caused by voltage-gated calcium and/or NMDAR channels (Andreasen and Nedergaard 1996; Magee and Johnston 1995). Thus the SK channel activation seen in these experiments (Fig. 7) may have mechanisms in common with the calcium spike-evoked SK channel activation shown in Fig. 1A.
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), we also repeated this type of experiment after blocking mGluRs. In the presence of the two mGluR blockers MPEP (10 µM) and LY341495 (50 µM), we obtained results that were indistinguishable from those shown in Fig. 7 (n = 6, P < 0.05, data not shown), indicating that mGlu receptors were not important for activating SK channels under these conditions.
These results are compatible with those of Ngo-Anh et al. (2005) and Bloodgood and Sabatini (2007) and also readily explain the lack of effect of apamin on the AHP following a train of synaptically evoked spikes during NMDAR blockade (Fig. 3). Thus if the activation of postsynaptic SK channels is dependent on NMDAR currents, directly or indirectly (Bloodgood and Sabatini 2007; Ngo-Anh et al. 2005), an apamin-induced enhancement of postsynaptic responses Fig. 7 can only occur in the absence of NMDAR blockade.
So far, we tested the roles of SK channels during synaptic transmission in the presence of NMDAR blockers (Fig. 3) or non-NMDAR blockers (Fig. 7). It is obviously of interest to examine the roles of SK channels also in the absence of any glutamate receptor blockers. Whereas previous studies tested the roles of SK channels in subthreshold postsynaptic events (Bloodgood and Sabatini 2007; Faber et al. 2005; Ngo-Anh et al. 2005; Stackman et al. 2002), we used high-frequency stimulation in st. radiatum, of sufficient strength to evoke a train of population spikes, to achieve optimal activation of SK channels and for comparison with somatically evoked spike trains and AHPs (Figs. 1, 2, 4, and 5) as well as postsynaptic responses in the presence of NMDAR blockers (Supplementary Fig. S4). The responses were measured in somatic field potential recordings (Supplementary Fig. S6), and the stimulation strength was adjusted so that the amplitudes of the population spikes were less than maximal to allow room for an increase. However, this stimulation paradigm tends to induce NMDAR-dependent plasticity in the non-NMDA glutamate receptors (LTP and LTD) (Bear 1996; Bliss and Lomo 1973). Therefore we allowed a long stabilization period for the LTP induction and expression to saturate (Bliss and Lomo 1973), thus achieving a stable baseline before apamin was applied. Under these conditions, bath application of apamin (100 nM) had no detectable effect on the train of population spikes or any other part of the postsynaptic responses (n = 6; Supplementary Fig. S6, A and E–H). In contrast, subsequent application of the Kv7/KCNQ/M-channel blocker XE-991 (10 µM; blue trace in Supplementary Fig. S6B), caused a clear increase in the first two population spikes (Supplementary Fig. S6, C and D). (Later in the train of population spikes, more complex effects of XE-991 were observed, including reduced population spike amplitudes, probably due to secondary effects of the enhanced initial spiking.) These effects of XE991, which we have previously found are purely postsynaptic under these conditions (Vervaeke et al. 2006), demonstrate that the population spikes could be increased by blockade of postsynaptic K+ channels under these conditions, thus indicating that the lack of an apamin effect was not due to trivial experimental factors.
The lack of an apamin effect may seem surprising in view of recent results indicating that dendritic spines of mouse CA1 pyramidal neurons contain SK channels that are specifically activated by Ca2+ influx through voltage-gated Ca2+ channels and/or NMDA channels in the spines (Bloodgood and Sabatini 2007; Ngo-Anh et al. 2005). Because Na+ action potentials backpropagate (bAPs) into the dendrites and spines (Bloodgood and Sabatini 2007; Ngo-Anh et al. 2005), they should help opening the voltage-gated Ca2+ and/or NMDA channels and hence activate the SK channels in the spines (Bloodgood and Sabatini 2007; Ngo-Anh et al. 2005). This would be expected to attenuate the EPSPs, thus reducing the population spike amplitudes. However, very recently, after the experiments of this study were completed, Lin et al. (2008) reported that LTP induction strongly downregulates the SK channel activity in the spines of mouse CA1 pyramidal neurons by removing SK2 channels from the postsynaptic membrane (PSD). This may explain the lack of apamin effect seen in Supplementary Fig. S6. According to this hypothesis, when LTP is induced by high-frequency stimulation during the long stabilization period, the SK channels in the spine are downregulated (Lin et al. 2008), precluding a subsequent effect apamin. In addition, as discussed in the following text, a local apamin effect in the spines or dendrites may have too weak impact in the soma to be detected there due to enhanced filtering by the dendritic cable during high-frequency discharge.
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DISCUSSION |
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) but are normally not activated by somatic Na+ spikes to an extent that appreciably influences the somatic response pattern, not even during high-frequency discharge.
These results may seem unexpected in view of the widespread idea that the main functions of SK channels in CA1 pyramidal cells and other neurons is action potential-triggered feedback control of excitability, including spike frequency adaptation and generation of afterhyperpolarizations (Faber and Sah 2003; Stackman et al. 2002; Stocker 2004; Stocker et al. 1999). Our results may also seem surprising because functional SK channels are evidently expressed at high density in the somatodendritic membrane of CA1 pyramidal cells and can generate a robust outward current (Sailer et al. 2002; Stocker et al. 1999). Furthermore, it is clear that normal action potentials in these cells cause a sufficient rise in intracellular calcium concentration to readily activate two other classes of Ca2+-activated K+ channels: the BK channels and the sAHP channels (Alger and Nicoll 1980; Faber and Sah 2003; Storm 1987a,b, 1990).
However, our results are fully compatible with other reports, including our previous studies showing no detectable SK channel contribution to the mAHP in CA1 pyramidal cells (Gu et al. 2005; Storm 1989). In particular, our results agree with previous evidence that the somatic mAHP in rat and mouse CA1 hippocampal pyramidal neurons are mainly caused by Kv7/M/KCNQ channels at depolarized potentials (approximately –60 mV) and by HCN/h channels at hyperpolarized potentials (approximately –80 mV) (Gu et al. 2005; Hu et al. 2007; Peters et al. 2005; Storm 1989); evidence that artificial enhancement of SK channel activity by application of an SK channel opener can effectively reduce the excitability of hippocampal pyramidal neurons (Pedarzani et al. 2001), although SK channels do not normally contribute appreciably to excitability control and adaptation in these cells (Gu et al. 2005; Storm 1989); and evidence that SK channels can be activated by calcium influx through NMDA-type glutamate receptor channels (Cai et al. 2004; Faber et al. 2005; Ngo-Anh et al. 2005; Stackman et al. 2002) or dendritic voltage-gated Ca channels (Bloodgood and Sabatini 2007; Cai et al. 2004; Ngo-Anh et al. 2005). This supports the possibility that SK channels may regulate, and even contribute to the expression of, LTP and LTD and perhaps other forms of synaptic plasticity that may underlie memory encoding (Cai et al. 2004; Faber et al. 2005; Ngo-Anh et al. 2005; Stackman et al. 2002).
Comparison with previous results on somatic excitability and AHPs
As already mentioned, our results appear to contradict several previous reports (Norris et al. 1998; Stackman et al. 2002; Stocker et al. 1999) and the widespread perception that SK channels are important generators of the mAHP in hippocampal pyramidal cells (Bildl et al. 2004; Bond et al. 1999, 2004; Bowden et al. 2001; Faber and Sah 2003; Melyan et al. 2002; Sah and Faber 2002; Sailer et al. 2002; Stocker 2004; Stocker et al. 2004).
It has been suggested (Stocker et al. 1999) that an SK channel component might have been overlooked in previous studies (Storm 1989) because bicuculline then was used routinely to block inhibitory synaptic input and various bicuculline salts have later been shown to be efficient SK channel blockers (Johnson and Seutin 1997). However, this explanation is clearly invalid for two reasons. First, in all our previous studies of AHPs (Pedarzani and Storm 1993, 1996; Storm 1987a,b, 1989), only the free base of bicuculline was always used for the explicit reason to avoid the risk of possible unknown side effects of the anions of bicuculline salts even before such side effects were discovered (Johnson and Seutin 1997). Therefore all salts of bicuculline that later were shown to block SK channels (Johnson and Seutin 1997) were always avoided in our previous studies; and bicuculline-free base, which was used in our studies, has later been shown to not block SK channels to any appreciable extent (Johnson and Seutin 1997) or block them by less than 10% (Khawaled et al. 1999) at the concentration used previously (5 µM) (Storm 1989). Second, we show in this study, like that of Gu et al. (2005), that an SK channel contribution could not be detected even in the complete absence of bicuculline.
Another tentative explanation for the discrepancies might be related to the use of different recording techniques and intracellular media. Whereas the early studies used KCl-filled sharp microelectrodes, which was commonly used at that time to achieve low, constant electrodes resistance for current- and voltage-clamp recording (Storm 1989; Williamson and Alger 1990), more recent studies that reported SK-channel involvement used whole cell recording with K-methylsulphate- or K-gluconate-based intracellular media (Stackman et al. 2002; Stocker et al. 1999). Because artificially high intracellular Cl– concentration has been reported by some groups to cause severe rundown of Ca2+-dependent K+ channel activity (Zhang et al. 1994); but see (Storm 1987a, 1989), one might worry that the early studies failed to detect a significant SK component because KCl suppressed SK channel activity. However, this hypothesis was disproved in the present study. Thus we tested a wide range of recording conditions (Table 1), including conditions identical to those used by Stocker et al. (1999) (Fig. 5) and Stackman et al. (2002) (whole cell; K-methylsulphate-based intracellular media) and found that a SK component could still not be detected (Table 1).
A more likely explanation for the discrepancy is the following: several ionic currents, in particular IM and Ca2+-dependent K+ currents in CA1 pyramidal cells, are prone to rundown during whole cell recording with a variety of intracellular media (see e.g., Fig. 2 in Zhang et al. 1994; Fig. 5C in Melyan et al. 2002; Fig. 6E in Sailer et al. 2002). It is therefore essential to study the time course of the effects of any kind of drug or manipulation as was done in our previous studies of AHPs (Pedarzani and Storm 1993, 1996; Sailer et al. 2002). This is particularly important in the absence of wash-out and recovery from SK channel blockade (Stackman et al. 2002; Stocker et al. 1999). Unfortunately, when time course plots are lacking (Stackman et al. 2002; Stocker et al. 1999), a steady rundown of channel activity, which occurs independently of drug application, might be mistaken for an effect of SK channel blockade.
Our results agree well with those of Yue and Yaari (2004), who showed that SK channel blockade by apamin failed to facilitate the somatic afterdepolarization (ADP) in these cells, whereas M-channel blockade had a strong effect. Our conclusion is also fully compatible with the finding that SK channel openers can enhance the mAHP in these cells (Pedarzani et al. 2001) as we also found (Fig. 5). The point is that the appearance of an SK component after artificial enhancement of SK channel activity does not indicate that these channels are involved in the mAHP under normal (drug-free) conditions. Our results are of course also compatible with the finding of Villalobos et al. (2004) that overexpression of SK1 or SK2 subunits in mice enhanced the mAHP in neocortical pyramidal cells. Furthermore, a recent study by Yoshida and Alonso (2007) showed that apamin had no significant effect on neuronal excitability, spike frequency adaptation, AHPs or ADPs in layer 5 pyramidal neurons in the rat entorhinal cortex, whereas M-channel blockade had clear effects on all these parameters (Yoshida and Alonso 2007). These findings closely parallel our results in rat and mouse hippocampal CA1–CA3 pyramidal cells in this and previous studies (Gu et al. 2005; Hu et al. 2007; Peters et al. 2005; Storm 1989) and suggest that the lack of SK channel impact on somatic spiking combined with strong M-channel effects applies to several, but not all (Fig. 6), classes of mammalian, cortical pyramidal cells.
An apamin-insensitive form of the human SK3-channel has been described (Wittekindt et al. 2004), and other toxin-insensitive forms may exist. However, it is unlikely that any of these, or any other Ca2+-activated current (Alger et al. 1994; Brown and Griffith 1983), are important for the mAHP because the mAHP is largely resistant to Ca2+-free medium and Ca2+ channel blockade (Gu et al. 2005; Storm 1989) as well as to the SK-channel blocker d-TC (Table 1 and Fig. 2B). Besides no orthologous form of the apamin-insensitive SK3 was found in rat (Wittekindt et al. 2004).
Although SK channels in CA1 pyramidal cells are readily activated by Ca2+ spikes evoked during blockade of Na+ and some K+ channels, as have been verified both in sharp electrode current-clamp (Fig. 1, A and B) and in whole cell voltage-clamp recordings (Sailer et al. 2002), they do not appear to be activated by the Ca2+ influx provided by somatic Na+ action potentials not even by high-frequency bursts. There seem to be at least four, not mutually exclusive, possible explanations. First, the SK channels in these cells may need a more massive Ca2+ influx (Cai et al. 2004) than either the BK or sAHP channels, which both are activated by a single spike (Lancaster and Nicoll 1987; Marrion and Tavalin 1998; Storm 1987a, 1990). Second, the SK channels, unlike BK channels, may not be efficiently co-localized with the voltage-gated Ca2+ channels that open during a normal Na+ spike (Lancaster and Nicoll 1987; Storm 1987b, 1990). Third, the sAHP channels may be more sensitive to [Ca2+]i than are SK channels, or fourth, may be targeted to a different subcellular compartment (Bloodgood and Sabatini 2007; Ngo-Anh et al. 2005).
Recently Bloodgood and Sabatini (2007) and Ngo-Anh et al. (2005) reported evidence that SK channels in dendritic spines are specifically activated by CaV2.3 (R-type) voltage-gated Ca2+ channels in the spines. Because Na+ action potentials backpropagate (bAPs) into the dendrites and spines (Bloodgood and Sabatini 2007; Koch and Zador 1993; Ngo-Anh et al. 2005), they open CaV2.3 channels and, hence, SK channels in the spines (Bloodgood and Sabatini 2007; Ngo-Anh et al. 2005). This would be expected to cause a local AHP, at least in the spine. However, because no apamin-sensitive AHP was observed in our somatic recording (Fig. 3), it seems likely that such local AHPs have too weak impact in the soma to be detected there due to filtering by the dendritic cable. Such filtering is probably strongly enhanced by the increased membrane conductance caused by other spike-evoked K+ channels, e.g., Kv7/M and delayed rectifier (DR) channels. In contrast, the Ca2+-spikes evoked in the presence of the K+ channels blocker TEA may cause sufficient depolarization of the somatodendritc membrane to open more voltage-gated Ca2+ channels, probably of several types, thus causing stronger SK channel activation. In addition, TEA (which partly blocks M and DR channels) will reduce the spike-evoked K+ conductance, thus reducing the filtering by the dendritic cable. These factors may explain why SK channels in CA1 pyramidal cells are readily activated by Ca2+ spikes and Ca2+ plateau potentials but not by Na+ spikes.
However, our results from CA1 pyramidal cells obviously do not preclude that SK channels can generate Na+ spike-evoked mAHPs in other types on neurons (Abel et al. 2004; Sah 1996; Sah and Faber 2002; Savic et al. 2001; Schwindt et al. 1988; Vogalis et al. 2003). On the contrary, we found that bursting pyramidal cells in the rat subiculum generate a prominent mAHP that is substantially reduced by SK channel blockade by apamin (Hu et al. 2004), and we have detected SK-dependent mAHPs also in rat neocortical pyramidal cells (Abel et al. 2004; Foehring et al. 1989; Hu and Storm, unpublished data) and cerebellar Purkinje cells (Cingolani et al. 2002; Hu and Storm, unpublished results) but not in rat CA3 pyramidal cells (Sharifullina et al. 2008). Thus it appears that the hippocampal CA1 and CA3 pyramidal cells differ from several other cell types by having a Kv7/KCNQ/M-channel-dependent mAHP rather than an SK-channel dependent mAHP (Gu et al. 2005; Storm 1989).
Impact of SK channel activation during synaptic transmission
While the SK channels could evidently be activated by NMDAR-mediated EPSPs (Fig. 7), we observed no detectable effect of SK channel blockade on EPSP-evoked spike trains and mAHPs in the presence of the NMDAR blocker D-AP5 (Fig. 3 and Supplementary Fig. S4). In the latter experiments, D-AP5 was used both to prevent NMDAR-dependent synaptic plasticity (LTP and LTD) and to test whether SK channels could be activated by synaptically evoked action-potentials as opposed to NMDAR-mediated EPSPs alone. Again these results indicate that SK channels in CA1 pyramidal cells do not substantially contribute to feed-back regulation or AHPs of somatic spikes, whether triggered by somatic or dendritic, non-NMDAR-mediated input.
However, under normal conditions, without NMDAR blockers, it seems likely that the SK channels are activated by EPSPs, whether spikes are triggered or not (Bloodgood and Sabatini 2007; Ngo-Anh et al. 2005). The spikes probably help to remove the magnesium block of the NMDARs, thus increasing their contribution to Ca2+ influx and SK activation. Nevertheless, the occurrence of action potentials may secondarily tend to mask the SK-channel effects. Thus once action potentials occur, they activate several voltage- and calcium-gated currents, including the M current (Fig. 3, A–C), which dominate the subsequent response and may thus swamp the effects of EPSP- and NMDAR-activated SK channels. Therefore the SK channel effect may, paradoxically, be relatively more important for subthreshold EPSPs than for EPSP-evoked spike trains.
How can our finding that apamin had no detectable effect on the train of population spikes (Supplementary Fig. S6) be reconciled with the recent finding that dendritic spines contain SK channels that are specifically activated by local voltage-gated Ca2+ channels and/or NMDA channels (Bloodgood and Sabatini 2007; Ngo-Anh et al. 2005)? One possibility is that SK channels in spines were downregulated by prior LTP induction as recently shown by Lin et al. (2008). However, in the experiments shown in Fig. 7, SK channels would be expected to undergo a similar downregulation due to massive opening of NMDA channels. A second possibility is that our method of stimulation was not able to evoked sufficient local dendritic depolarization to strongly activate the high-threshold R-type Ca2+ channels and/or NMDA channels that have been reported to be necessary for SK channel activation (Bloodgood and Sabatini 2007). To strongly activate these channels, there may be a need for near coincident and spatially close synaptic activation. Thus 15 to 20 synapses on the same dendrite branch need to be activated within a few milliseconds to evoke a substantial dendritic Ca2+ response (Losonczy and Magee 2006). Although bAPs are also able to activate R-type Ca2+ channels under certain conditions (Sabatini and Svoboda 2000), this has only been shown for very proximal spines in the proximal dendritic branches (Losonczy and Magee 2006; Sabatini and Svoboda 2000), whereas more distal branches are likely to receive a much more attenuated bAPs. Our stimulation electrode was positioned in the middle of st. radiatum and >100 µm laterally displaced from the main dendritic axis of the neurons from which we recorded the population spikes, to avoid direct activation of the dendrites. Thus it is possible that our stimulation protocol activated synapses that were too dispersed along the dendritic tree to strongly activate high-threshold Ca2+ channels and/or unblock NMDA channels and, hence, open dendritic SK channels.
Implications for SK channel functions
We conclude that the SK channels appear to have no obvious function in feed-back regulation of somatic spiking in CA1 hippocampal pyramidal cells in response to non-NMDAR-mediated excitatory input. First, the hypothesis that they underlie the sAHP has been undermined by several convergent results (Stocker et al. 2004; Vogalis et al. 2003). Second, several lines of evidence, including the present study, indicate that they also do not contribute appreciably to the somatic mAHP and excitability control (Gu et al. 2005; Peters et al. 2005; Storm 1989).
However, the presence of a robust SK current in the CA1 pyramidal cells suggests that these channels are of functional importance in these cells. Convergent evidence from several recent studies, including the present study, supports the hypothesis that SK channels can be activated by glutamatergic synaptic input through activation of NMDARs or voltage-gated Ca2+ channels in CA1 pyramidal dendrites (Bloodgood and Sabatini 2007; Ngo-Anh et al. 2005). Thus SK evidence indicates that channels in dendritic spines are directly activated by Ca2+ influx through NMDAR channels or Cav2.3 (R-type) Ca2+ channels (Faber et al. 2005; Ngo-Anh et al. 2005; Stackman et al. 2002; Tzounopoulos and Stackman 2003). Thereby they curtail the postsynaptic depolarization, rapidly reinstate the magnesium block of the NMDAR channels, and limit the Ca2+ influx. This SK channel-mediated negative feed-back control of postsynaptic Ca2+ may explain how these channel regulate the induction of synaptic plasticity (Faber et al. 2005; Ngo-Anh et al. 2005; Stackman et al. 2002; Tzounopoulos and Stackman 2003).
These ideas are supported by the findings that overexpression of SK2 channels in the hippocampus decreased synaptically evoked glutamatergic EPSPs, attenuated LTP induced by 50-Hz stimulation in hippocampal slices and drastically impaired learning and memory in two hippocampal-dependent behavioral tasks (Hammond et al. 2006). Recent data from Lin et al. (2008) also indicate that SK2 channels are themselves downregulated by LTP and that this modulation contributes to LTP expression in hippocampal CA1 cells. Thus they found that pairing of synaptically evoked EPSPs with backpropagating action potentials abolished SK2 channel activity, thus contributing to the enhanced EPSPs that characterize LTP expression. Indications that apamin can improve learning and memory retention and increase immediate early gene expression in the hippocampus of mice (Heurteaux et al. 1993; Messier et al. 1991) further support the idea that hippocampal SK channels have important functions in plasticity. Such functions may also be partly mediated by SK channels expressed in GABAergic interneurons (Savic et al. 2001; Zhang and McBain 1995).
That SK channels are also involved in other forms of neural plasticity is suggested by a recent study by Cai et al. (2007) that indicates that posttranslational downregulation of SK channel function in the distal dendrites of CA1 pyramidal cells contributes to deafferentation-induced reactive hyperexcitability. This new insight may open new possibilities for prevention or treatment of such conditions, e.g., in posttraumatic epilepsy. In addition, it is likely that SK channels also can regulate other forms of dendritic electrogenesis and may become engaged in different response patterns than those tested here.
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ACKNOWLEDGMENTS |
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1 The online version of this article contains supplemental data. 
Address for reprint requests and other correspondence: J. F. Storm, Dept. of Physiology at IMB and Centre for Molecular Biology and Neuroscience (CMBN), University of Oslo., PB 1103 Blindern, N-0317 Oslo, Norway (E-mail: jstorm{at}medisin.uio.no)
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), 3rd (
), and 4th (
) spike, and the steady-state firing frequency (
) during the 1-s spike train, plotted against the amplitude of the current pulse. The steady-state firing frequency was calculated as the average frequency during the last 400 ms of each current pulse; the instantaneous frequency of a given spike is defined as the inverse of the interspike interval (ISI) between that spike and the 1 preceding it. Apamin had no apparent effect on the firing frequencies, whereas XE991 increased the firing frequency (n = 5). C: averaged firing frequencies measured within 100-ms time windows during a 1-s, 0.15-nA current pulse. No significant effect of apamin was detected. In contrast, XE991 increased the early firing frequencies within the first 300 ms. D: average firing frequency of the 1-s spike train plotted against the current pulse amplitude. Apamin had no significant effect, whereas XE991 caused a strong increase in the average firing frequency in response to current pulses <0.5 nA.
