Dendritic calcium spike initiation and repolarization are controlled by distinct potassium channel subtypes in CA1 pyramidal neurons

Abstract

In CA1 pyramidal neurons of the hippocampus, calcium-dependent spikes occur in vivo during specific behavioral states and may be enhanced during epileptiform activity. However, the mechanisms that control calcium spike initiation and repolarization are poorly understood. Using dendritic and somatic patch-pipette recordings, we show that calcium spikes are initiated in the apical dendrites of CA1 pyramidal neurons and drive bursts of sodium-dependent action potentials at the soma. Initiation of calcium spikes at the soma was suppressed in part by potassium channels activated by sodium-dependent action potentials. Low-threshold, putative D-type potassium channels [blocked by 100 microM 4-aminopyridine (4-AP) and 0.5-1 microM alpha-dendrotoxin (alpha-DTX)] played a prominent role in setting a high threshold for somatic calcium spikes, thus restricting initiation to the dendrites. DTX- and 4-AP-sensitive channels were activated during sodium-dependent action potentials and mediated a large component of their afterhyperpolarization. Once initiated, repetitive firing of calcium spikes was limited by activation of putative BK-type calcium-activated potassium channels (blocked by 250 microM tetraethylammonium chloride, 70 nM charybdotoxin, or 100 nM iberiotoxin). Thus, the concerted action of calcium- and voltage-activated potassium channels serves to focus spatially and temporally the membrane depolarization and calcium influx generated by calcium spikes during strong, synchronous network excitation.

Fig. 1.

Initiation of calcium spikes. A, Complex spikes detected with dendritic patch-pipette recordings at three different distances from the soma. Each recording is from a different experiment, and the morphological reconstruction is from the recording in the middle panel (200 μm from the soma). In these recordings, fast action potentials decline in amplitude with activity before the generation of a larger amplitude complex spike (asterisks). These complex spikes are shown expanded at the right and consist of an initial small backpropagating action potential (arrows) that gives rise to one to two larger spikes. B, Synaptic responses of the same cells shown in A to a train of three shocks (arrowheads) applied to stratum radiatum in the presence of 10 μm bicuculline. These stimuli give rise to complex spikes similar to those evoked with dendritic current injection.C, The slow component of complex spikes (asterisks) is mediated by calcium channels because it is blocked reversibly by 500 μm Ni²⁺. Dendritic recording 224 μm from the soma. Insets show complex spikes expanded in time. Calibration (ininsets): 10 mV, 10 msec.

Fig. 2.

Calcium channel blockade eliminates complex spikes when applied locally to the dendrites but not the soma.A, A complex spike was evoked with dendritic current injection (500 pA through a single dendritic patch pipette 220 μm from the soma). B, Local pressure application of 5 mm Ni²⁺ to the somatic region caused repetitive firing of calcium spike bursts and increased the number of individual calcium spikes within a burst. C, The effects of Ni²⁺ were quickly reversed when the pressure application was interrupted. D, E, Calcium spikes were eliminated completely and reversibly when Ni²⁺ was applied to the dendrite, ∼100 μm distal to the recording pipette. There was an increase in excitability of the neuron upon washout of Ni²⁺. In schematic illustrations, the area targeted by the local application is indicated by gray ovals, and the direction of the local bath flow is indicated by a gray arrow.

Fig. 3.

Threshold for initiation of calcium spikes is lowest in the dendrites. A, Patch-pipette recordings made from the soma (thin traces) and apical dendrite (thick traces) 225 μm away. A calcium spike (asterisk) was initiated with dendritic current (top pair of traces) but not somatic current injection (bottom pair of traces). The calcium spike is shown expanded in the inset at the right of thetop pair of traces. Calibration (ininset): 10 mV, 20 msec. B, In the same cell as in A, the disparity between the thresholds of calcium spikes in response to somatic and dendritic current injection was primarily eliminated when action potentials were blocked in the presence of TTX. Isolated calcium spikes are shown expanded at theright of each pair of traces. Calibration (in inset): 10 mV, 20 msec.

Fig. 4.

Calcium spikes isolated in the presence of TTX are mediated in part by calcium channels localized near or in the soma.A, A presumed calcium spike was initiated with a 480 pA current pulse injected through the somatic patch pipette.B, C, Local pressure application of 5 mm Ni²⁺ raises reversibly the amount of current required to generate calcium spikes. D, Calcium spikes were blocked completely, even in response to strong depolarizations, when Ni²⁺ was applied to the soma and adjacent dendrites. In schematic illustrations, the area targeted by the local application is indicated by gray ovals, and the direction of the local bath flow is indicated by a gray arrow.

Fig. 5.

Potassium channels blocked by low concentrations of 4-AP increase the threshold for calcium spike initiation.A, Simultaneous patch-pipette recording from the soma (thin traces) and apical dendrite (thick traces) 160 μm away. A calcium spike (asteriskand inset) could be initiated with dendritic current injection (top pair of traces) but not with somatic current injection (bottom pair of traces).B, Same recordings as in A. Application of 100 μm 4-AP drastically reduced the threshold for calcium spike initiation in response to current injection in both the soma and dendrites. Insets show calcium spikes (asterisks) expanded in time. Also note the elimination of the afterhyperpolarization of backpropagating action potentials in 4-AP compared with normal saline. Calibration (ininsets): 20 mV, 10 msec. C, Voltage-clamp recordings from outside-out patches taken from the proximal apical dendrite of pyramidal cells. Left, A voltage command from −80 to +40 mV evoked both transient and steady-state outward currents in the presence of 1 μm TTX (Control). 4-AP (100 μm) blocked ∼16% of the fast transient component of the current and eliminated the steady-state component. Right, The effect of 100 μm 4-AP on outward currents was similar in the presence of calcium channel blockers Cd²⁺ (200 μm) and Ni²⁺ (50 μm).Insets show peak currents (asterisks) expanded in time. Calibration (in insets): 40 pA, 1 msec. Thick traces in insets are currents in the 4-AP condition. Patches were pulled from the apical dendrite 140 μm (left traces) and 130 μm (right traces) from the soma.

Fig. 6.

α-DTX-sensitive currents suppress calcium spike-driven burst generation at the soma and contribute to the afterhyperpolarization of sodium-dependent action potentials.A, In normal saline, bursting was not observed, even in response to a large step of current (top traces). However, the voltage and current thresholds for burst generation markedly decreased in the presence of 0.5 μm α-DTX (bottom traces). The first 200 msec of responses in each condition are shown expanded at the right.B, In the same cell, α-DTX reduced the voltage and current threshold for sodium-dependent action potential initiation only slightly and decreased the characteristic delay to firing.C, Superposition of the first sodium-dependent action potential in the traces shown in Breveals that 0.5 μm α-DTX increased the width of the first action potential (measured at half height) by 18% and reduced the fast afterhyperpolarization by ∼3 mV (82%). The weakened afterhyperpolarization in the DTX condition led to the generation of a second action potential in close succession to the first.

Fig. 7.

Calcium-activated potassium channels contribute to the repolarization of calcium spike bursts. A, Simultaneous patch-pipette recording from the soma (thin traces) and apical dendrite (thick traces) 250 μm away. A calcium spike (asterisk andinset) could be initiated with dendritic current injection (top pair of traces) but not with somatic current injection (bottom pair of traces).B, In the same cell, application of 250 μmTEA prolonged the burst of dendritic calcium spikes but only reduced slightly the threshold for calcium spike initiation in response to dendritic current injection. Calcium spikes still could not be initiated with somatic current injection in the presence of TEA.Insets show calcium spike bursts (asterisks) expanded in time. C, Voltage-clamp recordings from outside-out patches taken from the proximal apical dendrite of CA1 pyramidal cells. Left, A voltage command from −80 to +40 mV evoked both transient and steady-state outward currents in the presence of 1 μm TTX (Control). TEA (250 μm) did not affect the fast transient component of the current but blocked the majority of the steady-state outward current. Right, The effect of TEA was dependent on the activation of calcium channels, because no significant block of outward currents was observed in the presence of Cd²⁺ (200 μm) and Ni²⁺ (50 μm). Insets in both left and right panels show peak currents (asterisks) expanded in time. Calibration:left inset, 40 pA, 1 msec; right inset, 20 pA, 1 msec. Thick traces in insets are currents in the TEA condition. Patches were pulled from the apical dendrite 170 μm (left traces) and 84 μm(right traces) from the soma.

Fig. 8.

Long bursts of complex spikes evoked in the presence of calcium-activated potassium channel blockers are eliminated by 500 μm Ni²⁺.A, A complex spike was evoked by injecting current through a dendritic recording electrode 225 μm from the soma.B, Multiple bursts of complex spikes were evoked by the same stimulus in the presence of the 70 nm ChTX.C, D, Bursts of complex spikes evoked in the presence of ChTX were blocked reversibly with the addition of 500 μm Ni² to the bath.