An apamin-sensitive Ca2+-activated K+ current in hippocampal pyramidal neurons

Abstract

In hippocampal and other cortical neurons, action potentials are followed by afterhyperpolarizations (AHPs) generated by the activation of small-conductance Ca2+-activated K+ channels (SK channels). By shaping the neuronal firing pattern, these AHPs contribute to the regulation of excitability and to the encoding function of neurons. Here we report that CA1 pyramidal neurons express an AHP current that is suppressed by apamin and is involved in the control of repetitive firing. This current presents distinct kinetic and pharmacological features, and it is modulated differently than the apamin-insensitive slow AHP current. Furthermore, our in situ hybridizations show that the apamin-sensitive SK subunits are expressed in CA1 pyramidal neurons, providing a potential molecular correlate to the apamin-sensitive AHP current. Altogether, these results clarify the discrepancy between the reported high density of apamin-binding sites in the CA1 region and the apparent lack of an apamin-sensitive current in CA1 pyramidal neurons, and they may explain the effects of this toxin on hippocampal synaptic plasticity and learning.

Figure 1

Dark-field photomicrographs of sagittal and coronal sections through the rat hippocampal region hybridized with oligonucleotides specific for SK1 (A), SK2 (B), and SK3 (C) and control (D). (E and F) High magnification of the CA1 region, showing expression of SK2 in pyramidal neurons and in interneurons (arrowheads) in stratum radiatum (E) and in stratum oriens (F). CA1, CA3, pyramidal cell layer of the CA1 and CA3 fields; DG, granule cell layer of the dentate gyrus; h, hilus proper; p, CA1 pyramidal cell layer; r, stratum radiatum; o, stratum oriens. (Scale bars: A–D, 400 μm; E and F, 100 μm.)

Figure 2

Whole-cell recordings showing two distinct voltage-independent Ca²⁺-activated currents in CA1 pyramidal neurons. (A) In the absence of bicuculline (Left), a 100-ms depolarizing pulse to +10 mV in the presence of tetrodotoxin (0.5 μM) and tetraethylammonium (1 mM) elicits two distinct currents. In the presence of bicuculline (10 μM), the medium current (Medium) is blocked, whereas the slow AHP current, sI_AHP, persists unaffected (Right). (B) Representative example of the medium current measured in isolation after suppression of sI_AHP by intracellular application of 8CPT-cAMP (50 μM; Left), in comparison with sI_AHP measured in the presence of apamin (50 nM; Right). The decay time courses of both currents were fitted by mono-exponential functions (dotted lines). (C) The medium current is reversibly suppressed in nominally Ca²⁺-free ACSF containing 5 mM Mg²⁺. (D) The Ca²⁺ channel blocker Cd²⁺ (50 μM) produces a partially reversible suppression of the medium current. (E) Bar diagram summarizing the effects of Ca²⁺-free medium and Cd²⁺ (50–200 μM) on the medium current (left four bars) and sI_AHP (right three bars). Numbers of experiments are reported over the corresponding bars.

Figure 3

Pharmacological characterization of the bicuculline-sensitive current. At 50 nM, apamin (A) and scyllatoxin (D) selectively block the medium current and do not affect sI_AHP. The block of the medium current uncovers a Ca²⁺-dependent inward current. Similarly, in B apamin (50 nM) and in E scyllatoxin (50 nM) block the medium current measured in isolation in the presence of 8CPT-cAMP. (C) Dose–response curve for the block of the medium current by apamin. Data points were fit with a Langmuir isotherm, giving an IC₅₀ value of ≈480 pM and a Hill coefficient of 0.92. For each point n = 3–7; error bars are SEM. (F) Bar diagram summarizing the effects of apamin (50 nM), scyllatoxin (ScyTx; 50 nM), and tubocurarine (curare; 50 μM) on the medium current and sI_AHP.

Figure 4

Differential modulation of I_AHP and sI_AHP by neurotransmitters and second messengers. (A) The β-adrenergic receptor agonist isoproterenol (0.5 μM) suppresses completely sI_AHP and increases I_AHP. (B) The cholinergic agonist carbachol (2.5 μM) inhibits sI_AHP and produces a small decrease in I_AHP peak amplitude. On the right, the traces are displayed expanded and superimposed. (C) The decay of I_AHP and the rise time of sI_AHP, measured in the same cell in the presence of apamin, were fitted with monoexponential functions (dashed lines) and overlap to an extent variable from cell to cell: small overlap (Left), big overlap (Right). Depending on the degree of overlap, a suppression of sI_AHP would produce an apparent decrease in I_AHP peak amplitude. (D) Bar diagram summarizing the effects of isoproterenol, 8CPT-cAMP, and carbachol on I_AHP and sI_AHP.

Figure 5

The apamin-sensitive AHP plays a role in controlling the firing properties of CA1 pyramidal neurons. (A) Apamin (50 nM) reduces the medium (filled arrowhead) AHP, but not the slow AHP (open arrowhead), which is instead suppressed by 100 μM 8CPT-cAMP. The AHP was elicited by a spike burst in response to a 400-ms depolarizing current pulse generating always the same number of action potentials. Averages of five traces are shown. (B) Apamin (50 nM) produces an increase in the early firing frequency, without affecting spike frequency adaptation. 8CPT-cAMP instead suppresses adaptation. Same cell as in A. The depolarizing current pulses were 800 ms long and of constant strength. Membrane potential: −57 mV in A and B. (C) Action potentials evoked by a 100-ms current injection. Apamin (50 nM) modified the interspike trajectory and decreased the interval between second and third spike, as shown by the superimposed traces (Right). The reduction of medium AHP is also evident. Membrane potential: −55 mV. (D) Application of 8CPT-cAMP (100 μM) suppresses spike frequency adaptation. Addition of apamin (50 nM) causes a further reduction in the interspike intervals, suggesting that the apamin-sensitive AHP contributes to determine the firing rate. (E) Plot of the firing frequency at different interspike intervals for the cell displayed in D. The control (○) shows strong adaptation. 8CPT-cAMP (100 μM; ■) suppresses spike frequency adaptation and increases the number of spikes produced in response to the same stimulus strength. Apamin (50 nM; ●) further increases the instantaneous firing frequency, with a stronger effect on the first intervals. Depolarizing pulses: 70 pA, 800 ms. Membrane potential: −57 mV in D and E.