Small-conductance Ca2+-activated K+ channel type 2 (SK2) modulates hippocampal learning, memory, and synaptic plasticity

Abstract

Apamin-sensitive, small-conductance, Ca2+-activated K+ channels (SK channels) modulate neuronal excitability in CA1 neurons. Blocking all SK channel subtypes with apamin facilitates the induction of hippocampal synaptic plasticity and enhances hippocampal learning. In CA1 dendrites, SK channels are activated by Ca2+ through NMDA receptors and restrict glutamate-mediated EPSPs. Studies of SK channel knock-out mice reveal that of the three apamin-sensitive SK channel subunits (SK1-SK3), only SK2 subunits are necessary for the apamin-sensitive currents in CA1 hippocampal neurons. To determine the specific influence of SK2 channels on hippocampal synaptic plasticity, learning, and memory, we used gene targeting through homologous recombination in embryonic stem cells to generate transgenic mice that overexpress SK2 subunits by 10-fold (SK2+/T). In these mice, the apamin-sensitive current in CA1 neurons was increased by approximately fourfold, relative to wild-type (WT) littermates. In addition, the amplitude of synaptically evoked EPSPs recorded from SK2+/T CA1 neurons increased twice as much in response to SK channel blockade relative to EPSPs recorded from WT CA1 neurons. Consistent with this, SK2 overexpression reduced long-term potentiation after high-frequency stimulation compared with WT littermates and severely impaired learning in both hippocampus- and amygdala-dependent tasks. We conclude that SK2 channels regulate hippocampal synaptic plasticity and play a critical role in modulating mechanisms of learning and memory.

Figure 1.

SK2+/T mice specifically overexpress SK2 channels. a, Western blot reveals an ∼10-fold overexpression of SK2 channels in SK2+/T mice. Membranes were prepared from whole-brain homogenate from WT or SK2+/T mice. Increasing amounts of brain membrane proteins from SK2+/T mice (1.1, 3.3, 10, and 30 μg) were loaded onto the gel for semiquantitative analysis. b, Real-time PCR reveals that SK2 transcripts are overexpressed by ∼4.5-fold, whereas SK1 and SK3 transcripts are expressed at WT levels in SK2+/T mice. Error bars indicate SEM. *p < 0.05.

Figure 2.

SK2 overexpression increases the amplitude of the apamin-sensitive current. a, b, Representative whole-cell recordings of CA1 neurons from WT (a) and SK2+/T (b) mice before and after apamin (ap) are shown. Cells were voltage clamped at 55 mV, and tail currents were elicited after a 100 ms depolarizing step to +20 mV. Calibration: 200 pA, 0.5 ms. The insets show the subtracted apamin-sensitive current (calibration: 100 pA, 200 ms). c, Summary plot reveals that SK2 overexpression increases the amplitude of the apamin-sensitive current measured at 100 ms (ImAHP) by approximately fourfold but does not affect the apamin-insensitive current measured at 1 s (IsAHP). d, SK2 overexpression does not alter the decay kinetics of the ImAHP. Error bars indicate SEM. *p < 0.05.

Figure 3.

SK2 overexpression enhances the SK channel-mediated attenuation of the synaptically evoked glutamatergic EPSPs in CA1. An average EPSP waveform was derived from 20 EPSPs synaptically evoked in control condition in WT (a, inset) and SK2+/T (b, inset) mice before (thin line) and after (thick line) application of apamin. a, Summary plot of the EPSP amplitude of WT mice under control condition relative to the baseline period during washin of apamin (n = 5 cells). b, Summary plot of the EPSP amplitude of SK2+/T mice under control condition relative to the baseline period during washin of apamin (n = 7 cells). Calibration: 1 mV, 25 ms. The times of drug application are indicated by the horizontal bars. Error bars indicate SEM.

Figure 4.

SK2 overexpression impairs the induction of synaptic plasticity in a frequency-dependent manner. Field potentials were recorded from the CA1 region of WT (○) and SK2+/T (•) hippocampal slices. Representative traces from baseline (thin line) and at 50 min after tetanus (thick line) are displayed at the top of the figure from each experiment (calibration: 0.25 mV, 2 ms). a, LTP was induced with three tetani consisting of 100 pulses delivered at 50 Hz (0.1 Hz). Slices from SK2+/T mice exhibited significantly less LTP 30–40 min after 50 Hz stimulation than slices from WT mice (p = 0.018). b, SK2 overexpression did not disrupt 100 Hz LTP induced with three tetani consisting of 100 pulses delivered at 100 Hz (0.1 Hz). c, SK2 overexpression did not disrupt LTD induced with low-frequency stimulation of 1 Hz for 20 min. Error bars indicate SEM.

Figure 5.

SK2 overexpression does not alter basal synaptic transmission or presynaptic release mechanisms. a, fEPSP slope measures were plotted against the corresponding fiber volley amplitude evoked by stimulation of the Schaffer collaterals with increasing intensity. The inset depicts representative traces from an SK2+/T slice (calibration: 0.25 mV, 2 ms). For each genotype, fiber volley amplitude was a significant predictor for fEPSP slope (p < 0.001), and there was no genotype effect on synaptic transmission properties. b, SK2 overexpression did not alter the PPF ratio (p > 0.05). c, Decline in fEPSPs during 100 Hz stimulation was not altered by SK2 overexpression (p > 0.05). The inset depicts representative traces (calibration: 0.25 mV, 5 ms). Error bars indicate SEM. ○, WT; •, SK2+/T.

Figure 6.

SK2 overexpression impairs hippocampal-dependent learning and memory in the Morris water maze. a, b, Learning in the hippocampal-independent visible platform water maze task was not different between WT (○) and SK2+/T (•) mice as assessed by CDT with swim speed as a covariate (a), ANCOVA (p > 0.05), although SK2+/T mice swam slower than WT mice (b). c, d, Learning in the hippocampal-dependent hidden platform water maze is significantly impaired in SK2+/T mice relative to WT mice (c, p < 0.001), and there were no genotypic differences in swim speed in this version of the task (d, p > 0.05). e, During the final probe test 24 h after the last hidden platform training trial, SK2+/T mice spent significantly less time in the quadrant of pool relative to the WT mice (p < 0.001). f, Search ratios computed from final probe test data indicate that SK2+/T mice failed to show a spatial bias for the platform location; search ratios were equivalent to chance performance (p > 0.05) and were significantly lower than WT ratios (p < 0.001), indicating that SK2 overexpression severely restricts learning and remembering the location of the hidden platform. Search ratios were defined as the frequency of crossings through a circular zone around the platform location (f, inset) divided by the frequency of crossings into all four circular zones. g, h, Representative tracings of swim paths of two WT mice (g) and two SK2+/T mice (h) during the final probe test. Fourteen of the 17 WT mice exhibited platform search patterns similar to that depicted in g, which were characterized as accurate search. In contrast, 8 of the 14 SK2+/T mice exhibited search patterns similar to those depicted in h, which were characterized as random search (left trace) or egocentric search (right trace). Error bars indicate SEM.

Figure 7.

SK2 overexpression impairs contextual and cued fear conditioning. There was no genotypic difference in freezing behavior between WT and SK2+/T mice during the 5 min context pre-exposure session. During the context test 24 h after conditioning, both genotypes exhibited conditioned fear demonstrated by increased frequency relative to the pre-exposure session (p < 0.001), although percentage of freezing was significantly reduced in SK2+/T mice compared with WT controls (p < 0.001), indicating impaired contextual conditioning in SK2+/T mice. During the tone test, before the tone was presented (minute 1), there were no differences in percentage of freezing between genotypes (p > 0.05), indicating SK2 overexpression did not alter baseline freezing behavior. However, during the tone test, SK2+/T mice exhibited less freezing in response to the tone compared with WT mice (minute 2; p = 0.004), indicating that SK2 overexpression also impaired cued fear conditioning. Error bars indicate SEM. *p < 0.05.