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. 2016 Mar 2;115(3):1740–1748. doi: 10.1152/jn.01047.2015

Impact of calcium-activated potassium channels on NMDA spikes in cortical layer 5 pyramidal neurons

Tobias Bock 1, Greg J Stuart 1,✉
PMCID: PMC4809967  PMID: 26936985

Abstract

Active electrical events play an important role in shaping signal processing in dendrites. As these events are usually associated with an increase in intracellular calcium, they are likely to be under the control of calcium-activated potassium channels. Here, we investigate the impact of calcium-activated potassium channels on N-methyl-d-aspartate (NMDA) receptor-dependent spikes, or NMDA spikes, evoked by glutamate iontophoresis onto basal dendrites of cortical layer 5 pyramidal neurons. We found that small-conductance calcium-activated potassium channels (SK channels) act to reduce NMDA spike amplitude but at the same time, also decrease the iontophoretic current required for their generation. This SK-mediated decrease in NMDA spike threshold was dependent on R-type voltage-gated calcium channels and indicates a counterintuitive, excitatory effect of SK channels on NMDA spike generation, whereas the capacity of SK channels to suppress NMDA spike amplitude is in line with the expected inhibitory action of potassium channels on dendritic excitability. Large-conductance calcium-activated potassium channels had no significant impact on NMDA spikes, indicating that these channels are either absent from basal dendrites or not activated by NMDA spikes. These experiments reveal complex and opposing interactions among NMDA receptors, SK channels, and voltage-gated calcium channels in basal dendrites of cortical layer 5 pyramidal neurons during NMDA spike generation, which are likely to play an important role in regulating the way these neurons integrate the thousands of synaptic inputs they receive.

Keywords: cortex, dendrites, pyramidal neuron, SK channel

n-methyl-d-aspartate (NMDA) receptors are widely expressed in the central nervous system, where they influence the time course of excitatory postsynaptic potentials (EPSPs) and calcium influx during excitatory synaptic input. They are activated by glutamate (Buhrle and Sonnhof 1983; Furukawa et al. 2005), voltage gated as a result of magnesium block [Daw et al. (1993); Flatman et al. (1986); Mayer and Westbrook (1985); see Paoletti (2011) for review], and permeable to calcium as well as sodium and potassium (Dingledine et al. 1983; Garaschuk et al. 1996; Jahr and Stevens 1993; Schneggenburger et al. 1993). Because their open probability is dependent on the release of glutamate as well as the postsynaptic membrane potential, they are widely regarded as coincidence detectors of pre- and postsynaptic activity and known to play a crucial role in the induction and regulation of synaptic plasticity (Bliss and Collingridge 1993; Collingridge 1987; Nevian and Sakmann 2006; Tabone and Ramaswami 2012; Tsumoto 1990).

Another important aspect of NMDA channel activity is its involvement in dendritic excitability (Schiller et al. 2000). The opening of NMDA receptors is voltage dependent, and therefore, their activation can lead to the generation of regenerative events, called NMDA spikes, when activated in sufficient numbers by clustered—or even distributed—synaptic input (Larkum and Nevian 2008; Major et al. 2008; Milojkovic et al. 2004; Polsky et al. 2009; Schiller et al. 2000; Wei et al. 2001). In pyramidal neurons, NMDA spikes occur in basal, oblique, as well as apical tuft dendrites (Larkum et al. 2009; Losonczy and Magee 2006; Manita et al. 2011; Nevian et al. 2007; Schiller et al. 2000) and can play a role in spike timing-dependent synaptic plasticity (STDP) (Gordon et al. 2006; Takahashi and Magee 2009). Whereas NMDA spikes are local events (Larkum and Nevian 2008; Major et al. 2008; Rhodes 2006), the voltage response associated with these events can facilitate the generation of dendritic calcium spikes (Larkum et al. 2009) and is thought to have an impact on neuronal firing in vivo (Lavzin et al. 2012; Palmer et al. 2014; Polsky et al. 2009).

As NMDA spikes cause a large but localized increase in dendritic intracellular calcium, it is likely that they will be regulated by calcium-activated potassium channels. Previous work in cultured hippocampal pyramidal neurons has shown that small-conductance calcium-activated potassium channels (SK channels) help to repolarize plateau potentials evoked by dendritic glutamate photolysis (putative NMDA spikes), reducing their width and therefore, dendritic excitability, whereas large-conductance calcium-activated potassium channels (BK channels) had no impact (Cai et al. 2004). Whether SK channels perform a similar role in cortical pyramidal neurons during NMDA spikes is unknown. Here, we examine the impact of both SK and BK channels on NMDA spikes in basal dendrites of cortical layer 5 pyramidal neurons. We find that SK channels suppress the amplitude of NMDA spikes, while at the same time, enhance their generation due to complex interactions among NMDA receptors, SK channels, and R-type voltage-gated calcium channels. In contrast, BK channels had no impact on the generation of NMDA spikes or their properties.

METHODS

Animal preparation.

Wistar rats (4–6 wk old) were anesthetized by inhalation of isoflurane (2%) and decapitated, according to procedures approved by the Animal Ethics Committee of the Australian National University. The skull was opened and the brain removed and immediately transferred into ice-cold carbogenated artificial cerebrospinal fluid (ACSF; 125 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 25 mM NaHCO3, 3 mM KCl, 1.25 mM NaH2PO4, pH 7.2). During the slicing procedure, the MgCl2 concentration was increased to 5 mM to reduce cell excitability. The brain was cut along the midline, adhered to an angled (10–15°) slicing platform, and immediately re-submerged. Parasagittal brain slices (300 μm thick) were cut using a vibrating tissue slicer (Campden Instruments, Leicestershire, UK, or Leica Microsystems, Wetzlar, Germany) and transferred to a chamber containing ACSF at 35°C, where they were incubated for 40 min. Thereafter, they were held at room temperature (21°C) until required.

Electrophysiology.

All in vitro electrophysiological recordings were performed using an Olympus BX61 WI microscope, equipped with Dodt gradient contrast optics (Luigs & Neumann, Ratingen, Germany) and a fluorescent imaging system. Slices were continuously perfused with carbogenated ACSF at a rate of 2 ml/min at 34°C (±1°C). Borosilicate glass pipettes (inner diameter, 0.5 mm; outer diameter, 1 mm) were pulled by a computer-controlled electrode puller (Sutter Instrument, Novato, CA) and had open-tip resistances of 4–6 MΩ. Recording pipettes were filled with intracellular solution of the following composition: 130 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 4 mM Mg2+-ATP, 0.3 mM Na2-GTP, 10 mM Na2-phosphocreatine (pH set to 7.25 with KOH; osmolarity, 285 mosmol/kgH2O). The fluorescent dye Alexa 594 (5 μM) was included in the intracellular solution to visualize basal dendrites. Patch pipettes were electrically connected via a chlorided silver wire to a current-clamp amplifier (BVC-700A; Dagan, Minneapolis, MN) via the amplifier headstage, which was mounted on a remotely controlled micromanipulator (Luigs & Neumann). Cells were excluded from data analysis if the somatic resting membrane potential was more depolarized than −55 mV or if fluctuations in membrane potential >5 mV were observed at any time during the recording. Cells were also excluded if the somatic series resistance exceeded 20 MΩ or if the somatic series resistance changed by >10% during the recording.

Pharmacology.

Stock solutions of apamin and D(−)-2-amino-5-phosphonopentanoic acid (D-APV) (Tocris, Abingdon, UK) were prepared in water at a concentration of 500 μM and 50 mM, respectively, and kept at −20°C until required. For bath application, apamin and D-APV were diluted in ACSF to concentrations of 100 nM and 100 μM, respectively. Stock solutions of CdCl2 were dissolved in ACSF at a concentration of 200 mM and kept at 4°C until required (but not longer than 10 days). For bath application, CdCl2 was diluted in ACSF to a final concentration of 200 μM. Stock solutions of the R-type calcium channel blocker SNX482 and the BK channel blocker iberiotoxin (Tocris) were prepared in water at a concentration of 100 μM and stored at −20°C until required. For bath application, iberiotoxin was diluted to a final concentration of 100 nM in ACSF, whereas SNX482 was added to ACSF at a concentration of 300 nM. The alternative BK channel blocker Paxilline was added to ACSF at a concentration of 1 μM. During bath application of drugs, the effects of apamin and APV were observed within 2–6 min and 1–4 min, respectively. Therefore, only recordings 8 min after wash-in were used from the analysis.

Glutamate iontophoresis.

l-Glutamic acid (Tocris) was dissolved in ACSF to a concentration of 200 mM, and the pH was adjusted to 7.4 with NaOH. The fluorescent dye Alexa 594 (1 μM) was added to the glutamate solution to help visualize the application pipette under fluorescent light. Iontophoretic pipettes were made from borosilicate glass (outer diameter, 1 mm; inner diameter, 0.5 mm) and pulled to give a tip resistance of 120–140 MΩ when filled with this solution. The iontophoretic pipette was connected to the 1× gain headstage of an Axoclamp 2A current/voltage-clamp amplifier (Molecular Devices, Sunnyvale, CA). A backing current of −5 nA was maintained throughout the experiment to prevent spillage of glutamate from the iontophoretic pipette. The pipette was inserted into the slice and positioned under visual control in close proximity (within 5 μm) to a basal dendrite of the patched neuron at a location at least 120 μm from the soma, visualized under fluorescent light using a charge-coupled device camera (CoolSNAP EZ; Photometrics, Tucson, AZ). Increasing amounts of glutamate were applied using 10 ms iontophoretic current steps with an amplitude of 50–500 nA in steps of 50 nA. Plots of the iontophoretic current vs. the somatic response amplitude were fitted with a sigmoid and the iontophoretic current required to evoke NMDA spikes determined by taking the maximum of the differential of this function. We define this current as the NMDA spike threshold. Before pooling data across multiple cells, the iontophoretic current at NMDA spike threshold under control conditions in individual cells was set to zero.

Data acquisition and analysis.

Electrophysiological data were filtered at 10 kHz and acquired at 50 kHz by a Macintosh computer running AxoGraph X acquisition software (AxoGraph Scientific, Sydney, Australia) using an InstruTECH ITC-18 interface (HEKA Elektronik, Lambrecht/Pfalz, Germany). Data analysis was performed using AxoGraph X in combination with custom programs in MATLAB (MathWorks, Natick, MA) as well as Microsoft Excel (Microsoft, Redmond, WA). Prism 4 (GraphPad, San Diego, CA) was used for statistics and preparation of graphs. For paired data, Wilcoxon's nonparametric matched pairs test or a paired t-test (if a Gaussian distribution could be assumed) was used to test statistical significance. For multiple data sets, the Dunn's multiple comparison test was performed to determine statistical significance. Results are presented as average values ± SE.

RESULTS

Impact of SK channels on NMDA spikes.

To evoke NMDA spikes in basal dendrites of cortical layer 5 pyramidal neurons, glutamate was applied iontophoretically to basal dendrites (>120 μm from the soma) under visual control during somatic whole-cell recording. The amount of glutamate applied was increased linearly by increasing the iontophoretic current. At the soma, a linear increase in the amplitude of the voltage response at low iontophoretic current amplitudes was followed by a supralinear increase and a subsequent saturation of the voltage response at higher iontophoretic current amplitude (Fig. 1, A and B; n = 9). This supralinear behavior was abolished by the selective NMDA receptor antagonist APV (100 μM), showing that it requires NMDA receptor activation (Fig. 1, A and B; n = 9). Given the similarity of these observations with previous findings (Schiller et al. 2000), we conclude that the supralinear events evoked by glutamate iontophoresis in our experiments represent NMDA spikes. APV caused a small but not statistically significant reduction in the amplitude of somatic voltage responses evoked by iontophoretic currents below that required to generate NMDA spikes, whereas suprathreshold responses were decreased by almost 70% in the presence of the NMDA receptor antagonist (Fig. 1C; P < 0.0005; n = 9).

Fig. 1.

Fig. 1.

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Impact of apamin on N-methyl-d-aspartate (NMDA) spikes. A: somatic sub- and suprathreshold voltage responses during glutamate iontophoresis to a basal dendrite (180 μm from the soma) before (black) and after (green) bath application of 2-amino-5-phosphonopentanoic acid (APV). B: average amplitude of somatic voltage responses to increasing iontophoretic current (0 nA indicates NMDA spike threshold in control) before (black) and after (green) bath application of APV (n = 9). C: average amplitude of normalized somatic voltage responses to iontophoretic current below (Sub-threshold) and above (Super-threshold) NMDA spike threshold in control (black) and APV (green). Data normalized to the control suprathreshold response; gray lines represent individual data points. D: somatic sub- and suprathreshold voltage responses during glutamate iontophoresis to a basal dendrite (210 μm from the soma) before (black) and after (orange) bath application of apamin. E: average amplitude of somatic voltage responses to increasing iontophoretic current (0 nA indicates NMDA spike threshold in control) before (black) and after (orange) bath application of apamin (n = 12). F: average amplitude of normalized somatic voltage responses to iontophoretic current below (Sub-threshold) and above (Super-threshold) NMDA spike threshold in control (black) and apamin (orange). Data normalized to the control suprathreshold response. G and H: average amplitude of the iontophoretic current required to reach NMDA spike threshold (G) and NMDA spike half-width (H) before (black) and after (orange) apamin. *P < 0.05; **P < 0.01; ***P < 0.001.

We first investigated the impact of SK channels on NMDA spikes using the selective SK channel blocker apamin. To isolate the impact of SK channel activation on NMDA spikes, these experiments were carried out in the presence of iberiotoxin (100 nM) to block BK channels. Bath application of apamin (100 nM) had no effect on somatic voltage responses evoked by glutamate iontophoresis, which were subthreshold for NMDA spike generation (Fig. 1, D–F; P > 0.05; n = 12). Once the threshold for NMDA spike generation was reached, however, block of SK channels increased the amplitude of responses at the soma by, on average, 29% (Fig. 1F; P < 0.005; n = 12), indicating an important role for SK channels in regulating the amplitude of NMDA spikes. Interestingly, application of apamin also led to an increase in the iontophoretic current required to reach threshold for generation of NMDA spikes by 16% (Fig. 1, E and G; P < 0.0005; n = 12) while having no impact on the NMDA spike half-width (Fig. 1H; P > 0.05; n = 12). These data indicate that SK channels have opposing effects on dendritic excitability during NMDA spikes. On one hand, they act to reduce the amplitude of NMDA spikes, thereby decreasing the impact of NMDA spikes at the soma. On the other hand, they reduce the iontophoretic current threshold for eliciting NMDA spikes, therefore promoting their occurrence.

Location of SK channels.

To determine whether somatic or dendritic SK channels are involved in regulating NMDA spike amplitude and threshold, we analyzed the effect of apamin on the passive membrane properties at the soma. Apamin was found to have no impact on the somatic resting membrane potential or input resistance (Fig. 2, A–C). Furthermore, apamin had no impact on somatic voltage responses of similar amplitude to NMDA spikes generated by current injection via the somatic recording pipette (Fig. 2D). As SK channels activate within 50–100 ms after a somatic action potential (AP) (Schwindt et al. 1988), we compared the average membrane potential, 50–200 ms after the onset of these current steps under control conditions and after bath application of apamin, but no changes were observed (Fig. 2E). In contrast, the afterhyperpolarization, 50–200 ms after AP onset, was reduced significantly by bath application of apamin in these experiments (Fig. 2, F and G; P < 0.001; n = 12). These data indicate that somatic SK channels are activated by the calcium influx during APs but not by subthreshold somatic depolarizations similar to that observed during NMDA spikes. We conclude that the SK channels responsible for the observed changes to NMDA spike threshold and amplitude are located distal to the soma, presumably in the basal dendrites of layer 5 pyramidal neurons.

Fig. 2.

Fig. 2.

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Impact of somatic small-conductance calcium-activated potassium channels. A: somatic membrane potential during 300 ms current steps (ranging from −150 to 250 pA) used to calculate the input resistance. B: average resting membrane potential before (black) and after (orange) bath application of apamin (n = 12). C: average input resistance before (black) and after (orange) bath application of apamin (n = 12). D: somatic membrane potential during a 300-ms, 350-pA current step that depolarized the cell soma to a similar extent as glutamate iontophoresis in the same cell. E: average depolarization of the cell within 50–200 ms of a 300-ms current step, as shown in D, before (black) and during (orange) bath application of apamin (n = 12). F: medium afterhyperpolarization potential (AHP) after a single action potential (AP) evoked by a 300-ms current step before (black) and after (orange) bath application of apamin (AP is truncated). G: average medium AHP after a single AP evoked by a 300-ms current step before (black) and after (orange) bath application of apamin (n = 12). ***P < 0.001.

Role of voltage-gated calcium channels.

We next investigated the source of calcium influx driving SK channel activation during NMDA spikes. One possibility is that the opposing effects of SK channel block on NMDA spike amplitude and threshold are the result of calcium influx via different sources—NMDA receptors and dendritic voltage-gated calcium channels. To test this idea, the impact of apamin on NMDA spikes was investigated in the presence of the nonselective voltage-gated calcium channel blocker cadmium (200 μM), which was added to the bath solution. In the presence of cadmium, application of apamin led to a similar increase in NMDA spike amplitude (Fig. 3, A–C; P < 0.005; n = 11); however, an increase in iontophoretic current threshold in apamin was no longer observed (Fig. 3D; P > 0.05; n = 11). As was the case under control conditions, in the presence of cadmium, apamin had no significant effect on NMDA spike half-width (Fig. 3E; P > 0.05; n = 11).

Fig. 3.

Fig. 3.

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Role of voltage-gated calcium channels. A: somatic sub- and suprathreshold voltage responses to increasing glutamate iontophoresis to a basal dendrite (150 μm from the soma) before (black) and after (orange) bath application of apamin in the presence of cadmium (200 μM). B: average amplitude of somatic voltage responses to increasing iontophoretic current (0 nA indicates NMDA spike threshold in control) before (black) and after (orange) bath application of apamin in the presence of cadmium (n = 11). C: average amplitude of normalized somatic voltage responses to iontophoretic current injections below (Sub-threshold) and above (Super-threshold) NMDA spike threshold before (black) and after (orange) apamin in the presence of cadmium (n = 11). Data normalized to the control suprathreshold response. D and E: average amplitude of the iontophoretic current necessary to reach NMDA spike threshold (D) and NMDA spike half-width (E) before (black) and after (orange) apamin in the presence of cadmium. F: somatic sub- and suprathreshold voltage responses to increasing glutamate iontophoresis to a basal dendrite (150 μm from the soma) before (black) and after (orange) bath application of apamin in the presence of SNX482. G: average amplitude of somatic voltage responses to increasing iontophoretic current (0 nA indicates NMDA spike threshold in control) before (black) and after (orange) bath application of apamin in the presence of SNX482 (n = 5). H: average amplitude of normalized somatic voltage responses to iontophoretic current injections below (Sub-threshold) and above (Super-threshold) NMDA spike threshold before (black) and after (orange) apamin in the presence of SNX482. I and J: average amplitude of the iontophoretic current required to reach NMDA spike threshold (I) and NMDA spike half-width (J) before (black) and after (orange) apamin in the presence of SNX482. K: somatic sub- and suprathreshold voltage responses to increasing glutamate iontophoresis to a basal dendrite (160 μm from the soma) before (black) and after (red) bath application of SNX482. L: average amplitude of somatic voltage responses to increasing iontophoretic current (0 nA indicates NMDA spike threshold in control) before (black) and after (red) bath application of SNX482 (n = 8). M: average amplitude of normalized somatic voltage responses to iontophoretic current injections below (Sub-threshold) and above (Super-threshold) NMDA spike threshold before (black) and after (red) bath application of SNX482. N and O: average amplitude of the iontophoretic current required to reach NMDA spike threshold (N) and NMDA spike half-width (O) before (black) and after (red) bath application of SNX482. *P < 0.05; **P < 0.01.

As previous work indicates that calcium influx through R-type voltage-gated calcium channels is required for activation of SK channels in dendrites and spines of pyramidal neurons (Bloodgood and Sabatini 2008; Giessel and Sabatini 2011; Jones and Stuart 2013), we investigated the role of R-type voltage-gated calcium channels using the blocker SNX482. In the presence of the SNX482 (300 nM), application of apamin still increased NMDA spike amplitude (Fig. 3, F–H; P < 0.05; n = 5) but had no significant impact on NMDA spike threshold (Fig. 3I; P > 0.05; n = 5) or half-width (Fig. 3J; P > 0.05; n = 5). Finally, we investigated the impact of SNX482 alone. Bath application of SNX482 (300 nM) caused no change in NMDA spike amplitude (Fig. 3, K–M), however increased the iontophoretic current required to elicit NMDA spikes by a similar magnitude to that observed during apamin application (Figure 3N; P < 0.05; n = 8; cf. Fig. 1G). SNX482 had no impact on NMDA spike half-width (Fig. 3O; P > 0.05; n = 8).

Together, these results show that blocking R-type voltage-gated calcium channels can both occlude and mimic the impact of apamin on NMDA spike threshold but has no impact on NMDA spike amplitude. This suggests that the impact of SK channels on NMDA spike threshold requires calcium influx through R-type voltage-gated calcium channels. In contrast, the effect of SK channels on NMDA spike amplitude was independent of the activation of voltage-gated calcium channels and presumably requires calcium influx through the NMDA receptor itself. The half-width of NMDA spikes was unaffected by apamin, cadmium, and SNX482, indicating that SK channels do not regulate the duration of NMDA spikes independent of the calcium source.

Impact of BK channels on NMDA spikes.

Finally, we next investigated the impact of BK channels on NMDA spikes using the BK channel blocker iberiotoxin. BK channels have been shown to modulate the width and generation of dendritic calcium spikes in the apical dendrites of layer 5 pyramidal neurons (Benhassine and Berger 2009) and might therefore also be expected to modulate NMDA spikes. As a positive control, we first investigated the impact of iberiotoxin (100 nM) on APs evoked by somatic current injection. Consistent with previous findings (Sah and Davies 2000; Shao et al. 1999), block of BK channels by iberiotoxin increased somatic AP half-width (Fig. 4, A and B; P < 0.05; n = 15), indicating that BK channels are involved in repolarization of somatic APs. In contrast to its effects on somatic APs, bath application of iberiotoxin (100 nM) had no impact of NMDA spike threshold, amplitude, or half-width (Fig. 4, C–G; n = 5). These experiments were repeated using the alternative BK channel blocker Paxilline (1 μM), yielding identical results (n = 5; data not shown). Therefore, we conclude that BK channels do not have a significant impact on NMDA spikes in basal dendrites of cortical layer 5 pyramidal neurons.

Fig. 4.

Fig. 4.

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Impact of large-conductance calcium-activated potassium channels (BK channels) on NMDA spikes. A: somatic AP before (black) and after (purple) block of BK channels by bath application of iberiotoxin (IbTX). B: average half-width of somatic APs before (black) and after (purple) bath application of iberiotoxin (n = 15). C: somatic sub- and suprathreshold voltage responses to increasing glutamate iontophoresis to a basal dendrite (180 μm from the soma) before (black) and after (purple) bath application of iberiotoxin. D: average amplitude of somatic voltage responses to increasing iontophoretic current (0 nA indicates NMDA spike threshold in control) before (black) and after (purple) bath application of iberiotoxin (n = 15). E–G: average NMDA spike amplitude (E), threshold (F), and half-width (G) in control (black) and after (purple) bath application of iberiotoxin (n = 15). *P < 0.05.

DISCUSSION

The results presented in this study show that SK channels regulate NMDA spike amplitude and threshold, whereas BK channels do not. Application of apamin increased NMDA spike amplitude, indicating that under control conditions, SK channels act to reduce the amplitude of NMDA spikes and therefore, decrease dendritic excitability. In contrast, we also observed that apamin increased the iontophoretic current required to generate NMDA spikes, indicating that under control conditions, SK channels reduce NMDA spike threshold, which would be expected to increase dendritic excitability. We define NMDA spike threshold here, not in terms of the membrane voltage but rather, as the amount of glutamate required to activate sufficient numbers of NMDA receptors to trigger an NMDA spike. This counterintuitive effect of apamin was blocked by the presence of cadmium and SNX482 and could be mimicked by SNX482, suggesting a role of R-type calcium channels (Fig. 3). These observations are consistent with earlier work showing that application of cadmium plus nickel increases the laser intensity required for NMDA spike generation during glutamate uncaging (Major et al. 2008), although in these experiments, this effect was attributed to a direct action on voltage-activated calcium channels rather than via SK channels, as we show here.

Given that SNX482 can inhibit A-type potassium channels (Kimm and Bean 2014), whereas cadmium can shift their voltage dependence to more depolarized potentials (Song et al. 1998; Wickenden et al. 1999), this raises the question of whether the impact of cadmium and SNX482 on NMDA spikes is via A-type potassium channels rather than SK channels. We think this is unlikely, as blocking A-type potassium channels enhances putative NMDA spikes in hippocampal pyramidal neurons (Cai et al. 2004) and so would be expected to decrease NMDA spike threshold, not to increase it. Furthermore, the impact of apamin on NMDA spike threshold was blocked by both cadmium and SNX482. As an interaction between R-type calcium channels and SK channels has previously been reported during both EPSPs evoked by glutamate uncaging and APs (Bloodgood and Sabatini 2008; Giessel and Sabatini 2011; Jones and Stuart 2013), we conclude that the effect of SNX482 and cadmium on NMDA spike threshold is most likely due to block of R-type voltage-gated calcium channels. In conclusion, our experiments reveal complex interactions among NMDA receptors, SK channels, and voltage-gated calcium channels in basal dendrites of cortical layer 5 pyramidal neurons during NMDA spike generation, which have opposing effects on dendritic excitability. In contrast, BK channels did not influence NMDA spike amplitude, width, or threshold.

The observation that SK channels suppress the amplitude of NMDA spikes, thereby reducing dendritic excitability, is consistent with earlier work in CA1 pyramidal neurons in slice cultures during putative NMDA spikes generated by photolysis of glutamate (Cai et al. 2004). Interestingly, in our experiments, SK channels only influenced the amplitude of NMDA spikes and did not influence their duration. This observation contrasts with these earlier findings, where SK channels in CA1 pyramidal neurons were found to regulate the duration of putative NMDA spikes but not their amplitude (Cai et al. 2004). Consistent with this earlier work, we found that BK channels did not influence the amplitude, width, or threshold of NMDA spikes (Fig. 4). This observation is somewhat surprising, given that BK channels reduce the duration of dendritic calcium spikes in the apical dendrites of layer 5 pyramidal neurons (Benhassine and Berger 2009), and suggests that BK channels are either not present in basal dendrites of cortical layer 5 pyramidal neurons or, if present, are not activated by NMDA spikes.

The finding that SK channels decrease the threshold for NMDA spike generation, thereby increasing dendritic excitability, is counterintuitive, as the opening of potassium channels would be expected to have had the opposite effect. This paradoxical action of dendritic SK channels was dependent on activation of voltage-gated calcium channels, as it was absent in the presence of cadmium and SNX482 and could be mimicked by blocking R-type channels. Whereas the main charge carrier during NMDA spikes is the NMDA receptor itself, the threshold for generation of NMDA spikes is known to depend on activation of dendritic voltage-gated calcium channels (Schiller et al. 2000). An increase in the availability of these channels at resting membrane potentials would be expected to decrease NMDA spike threshold and may explain the counterintuitive impact of apamin on NMDA spike threshold. Constitutive activation of SK channels at resting membrane potentials would be expected to hyperpolarize basal dendrites, removing inactivation of voltage-gated calcium channels, and thereby increase their availability for activation. This would facilitate the generation of NMDA spikes and may explain why the blocking of SK channels with apamin increases NMDA spike threshold. Although we did not see a significant effect of apamin on the somatic resting membrane potential or input resistance (Fig. 2), it is possible that blocking SK channels impacts the membrane potential in distal basal dendrites or dendritic spines. Whatever the mechanism, the observation that apamin had no impact on somatic responses of similar amplitude to NMDA spikes suggests that the SK channels involved in modulating NMDA spikes are located in the basal dendrites rather than at the soma and possibly in dendritic spines.

The impact of apamin on the amplitude of NMDA spikes was preserved in the presence of voltage-gated calcium channel blockers (Fig. 3). These data indicate that the calcium source for this effect is via the NMDA receptor itself, consistent with recent evidence, indicating that during EPSPs, SK channels are activated solely by calcium influx through NMDA receptors (Wang et al. 2014). In contrast, SK channels activated by back-propagating APs in basal dendrites of cortical layer 5 pyramidal neurons are activated by calcium influx through R-type voltage-gated calcium channels (Jones and Stuart 2013), as observed during EPSP-like events generated by glutamate uncaging (Bloodgood and Sabatini 2008; Giessel and Sabatini 2011).

We conclude from our data that the opposing effects of SK channels on NMDA spikes depend on different calcium sources. When SK channels are activated by calcium influx through R-type voltage-gated calcium channels, this lowers the threshold for NMDA spikes, increasing dendritic excitability. In contrast, when SK channels are activated by calcium influx through NMDA receptors, this reduces the amplitude of NMDA spikes, reducing dendritic excitability. How this differential activation of SK channels occurs is unclear but may result from colocalization of different populations of SK channels with NMDA receptors and R-type voltage-gated calcium channels in separate microdomains.

Interestingly, SK channel block did not lead to a significant change in the amplitude of voltage responses subthreshold for NMDA spike generation (Fig. 1F). This observation in cortical layer 5 pyramidal neurons is in contrast to earlier work, showing that SK channels act to limit the amplitude of subthreshold synaptic responses in pyramidal neurons from the hippocampus and amygdala (Bloodgood and Sabatini 2008; Faber et al. 2005; Ngo-Anh et al. 2005), but similar to a recent study in mouse prefrontal cortex, where small (<5 mV) responses to glutamate uncaging were unaffected by apamin (Seong et al. 2014). These observations suggest that in cortical pyramidal neurons, small synaptic responses do not activate sufficient NMDA receptors for SK channel activation. Brain region-specific differences in the properties of SK channels or NMDA receptors or their colocalization may account for this difference. As indicated above, differences between cortical and hippocampal pyramidal neurons are also visible when comparing the impact of SK channel activation on suprathreshold responses. In CA1 hippocampal pyramidal neurons, SK channels limit the duration of putative NMDA spikes without influencing their amplitude (Cai et al. 2004), whereas in cortical layer 5 pyramidal neurons, SK channels reduce their amplitude but not their width (Figs. 1 and 3). Since the time course of suprathreshold responses at the soma in both studies is similar, it can only be speculated that there are differences in the properties or distribution of the channels involved. For example, SK channels in cortical pyramidal neurons may be more tightly colocalized with their calcium source and therefore, activate faster, thereby having a greater impact on NMDA spike peak. Differences in colocalization of SK channels and their calcium source between different brain regions and subcellular compartments have been shown in multiple studies. For example, in the soma of CA1 hippocampal neurons, SK channels are tightly colocalized with L-type calcium channels (Marrion and Tavalin 1998), whereas in their spines, they are activated by R-type calcium channels and NMDA receptors (Bloodgood and Sabatini 2008; Ngo-Anh et al. 2005). In other brain regions, SK channels are activated specifically by calcium influx through acetylcholine receptors or T-type calcium channels [Cueni et al. (2008); Oliver et al. (2000); see Fakler and Adelman (2008) for review]. Whereas immunohistochemical studies suggest that SK2 channels are expressed at the soma and proximal basal dendrites of layer 5 pyramidal neurons and SK1 in the more distal apical dendrites (Sailer et al. 2002), the nature of the subcellular colocalization of different SK channels with their calcium source(s) for activation is unknown. Aside from colocalization, differences in intracellular calcium buffering (Anwar et al. 2012) and the specific subtypes of voltage-gated calcium channels activated during NMDA spikes may also account for differences in the impact of SK channels on NMDA spikes in hippocampal and cortical pyramidal cells.

The impact of SK channels on NMDA spikes would also be expected to play a role in synaptic plasticity. Indeed, a number of studies indicate an important role of SK channels in regulating synaptic plasticity (Faber et al. 2005; Harvey-Girard and Maler 2013; Hopf et al. 2010; Ohtsuki et al. 2012). Given that SK channels influence NMDA spike amplitude rather than width, during STDP, it seems likely that SK channels will impact STDP strength, rather than the time window for STDP induction in cortical pyramidal neurons (Kampa et al. 2006; Letzkus et al. 2006; Schiller and Schiller 2001). In contrast, in hippocampal pyramidal neurons, where SK channels regulate the duration of putative NMDA spikes, the opposite is likely to be the case.

Finally, the observation that SK channels located in basal dendrites of layer 5 pyramidal neurons cause opposing, but not necessarily equal, effects on NMDA spikes makes it difficult to predict their impact on neuronal output. Furthermore, it is unclear how these effects of SK channels on NMDA spikes interact with dendritic calcium spikes and back-propagating APs—an interaction that will influence their impact on AP output. The situation is complicated further by the fact that NMDA spikes themselves can facilitate the initiation of dendritic calcium spikes in the apical dendrites of cortical layer 5 pyramidal neurons (Larkum et al. 2009). This raises the broader issue of how SK channels impact neuronal excitability in general. Does this occur primarily through regulating dendritic excitation or via the well-described impact of SK channels on regulating the AP afterhyperpolarization at the soma? Future studies will be required to dissect these aspects of SK channel function on cortical excitability, ideally in vivo in awake, behaving animals.

GRANTS

This work was supported by Australian National University and the Australian Research Council Centre of Excellence for Integrative Brain Function.

DISCLOSURES

The authors declare no competing financial interests.

AUTHOR CONTRIBUTIONS

Author contributions: T.B. and G.J.S. conception and design of research; T.B. performed experiments; T.B. analyzed data; T.B. and G.J.S. interpreted results of experiments; T.B. prepared figures; T.B. and G.J.S. drafted manuscript; T.B. and G.J.S. edited and revised manuscript; T.B. and G.J.S. approved final version of manuscript.

ACKNOWLEDGMENTS

Present address of T. Bock: Dept. of Neuroscience, Columbia University Medical Center, Kolb Annex, 1051 Riverside Dr., New York, NY 10032.

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