Prolonged activation of Ca2+-activated K+ current contributes to the long-lasting refractory period of Aplysia bag cell neurons

Abstract

Stimulation of the bag cell neurons of Aplysia activates several biochemical pathways, including protein kinase C (PKC), and alters their excitability for many hours. After an approximately 30 min afterdischarge, these neurons enter an approximately 18 hr inhibited state during which additional stimulation fails to evoke discharges. In vivo, this refractory period limits the frequency of reproductive behaviors associated with egg laying. We have now examined the role of Ca2+-activated K+ (BK) currents in the refractory period. Outward currents gated by both intracellular Ca2+ and depolarization, with pharmacological characteristics of BK currents, were recorded in isolated bag cell neurons. These currents were enhanced by the BK channel activators phloretin and 1,3-dihydro-1-[2-hydroxy-5-(trifluoro-methyl)phenyl]-5-trifluoromethyl-2H-benzimidazol-2-one and inhibited by the BK blocker paxilline. The BK component of K+ current was enhanced by 12-O-tetradecanoyl-phorbol-13-acetate, an activator of PKC, and this effect was blocked by sphinganine and PKC(19-36), inhibitors of PKC in bag cell neurons. To test whether the BK current is altered during the refractory period, intact clusters were stimulated to afterdischarge, and neurons were isolated after the clusters had entered the refractory period. Compared with unstimulated cells, current density was almost doubled in refractory neurons. This increase in current was inhibited by preincubating clusters in sphinganine. Treatment of refractory clusters with paxilline significantly restored the ability of stimulation to evoke afterdischarges. Conversely, application of phloretin to previously unstimulated clusters inhibited the onset of afterdischarges. These results indicate that a prolonged increase in BK channel activity contributes to the prolonged refractory period of the bag cell neurons.

Fig. 1.

Current–voltage relationships for whole-cell K⁺ currents of isolated bag cell neurons. Currents were recorded with Ca²⁺-free intracellular solutions or solutions containing 1 μm Ca²⁺ and evoked by depolarizing the membrane from a holding potential of –40 mV to test potentials from –40 to +60 mV. Peak K⁺currents were normalized by cell capacitance (n = 9).

Fig. 2.

Effect of paxilline on Ca²⁺-activated K⁺ currents in bag cell neurons. A, Mean current density–voltage relationships for cells dialyzed with 1 μmCa²⁺ in the intracellular solution before and after exposure to 10 μm paxilline (n = 5). Also shown are representative current traces and the difference currents obtained by subtracting traces from a paxilline-treated cell from the control traces obtained before paxilline exposure. B, Mean current density–voltage relationships and representative current traces for cells dialyzed with Ca²⁺-free intracellular solution before and after exposure to 10 μm paxilline (n = 5).

Fig. 3.

Effect of phloretin on Ca²⁺-activated K⁺ currents in bag cell neurons. Mean current density–voltage relationships are shown for cells dialyzed with 1 μm Ca²⁺ in an intracellular solution before and after exposure to 100 μm phloretin (n = 5). Also shown are representative current traces and the difference currents obtained by subtracting control traces from those obtained after phloretin exposure.

Fig. 4.

K⁺ currents are increased by TPA in bag cell neurons. A, Mean values of peak current density for five cells dialyzed with 1 μmCa²⁺ before and after treatment with 100 nm TPA. Also shown are representative currenttraces and the difference currents obtained by subtracting control traces from those obtained after TPA exposure.B, Mean values of peak current density and representative traces for cells dialyzed with 1 μm Ca²⁺ before and after treatment with 4α-phorbol (100 nm; n = 5).

Fig. 5.

Inhibitors of PKC prevent the increase in K⁺ current produced by TPA. Mean values of peak current density and representative current traces are shown for cells dialyzed with 1 μmCa²⁺ before and after treatment with 100 nm TPA in the presence of sphinganine (10 μm;n = 5; A) and after dialysis with the pseudosubstrate inhibitor PKC_19–36 (50 μm; n = 5; B).

Fig. 6.

TPA enhances Ca²⁺-activated K⁺ currents in bag cell neurons. A, Mean values of peak current density and representative currenttraces showing the lack of effect of 100 nmTPA in cells dialyzed with a Ca²⁺-free intracellular solution (n = 5). B, Mean values of peak current density and representative current tracesfor cells dialyzed with 1 μm Ca²⁺before and after treatment with 100 nm TPA and subsequent application of 10 μm paxilline (n = 5). Also shown are the difference currents.

Fig. 7.

K⁺ currents are enhanced after an afterdischarge in the bag cell neurons. A, Mean current density–voltage relationships for cells isolated from unstimulated clusters (Control, n = 9) and from those that had been stimulated to afterdischarge at least 3 hr before recording (n = 9). Also shown are data for cells stimulated in the presence of 25 μm sphinganine applied 15 min before stimulation (n = 9).B, Comparison of the effect of paxilline (10 μm) on K⁺ currents recorded in cells isolated from unstimulated clusters (n = 9) and in those that had been stimulated to afterdischarge (n= 9). Paxilline reduced the currents to a significantly greater extent in the cells that had undergone afterdischarge.

Fig. 8.

Inhibitors and activators of BK channels influence the refractory state of bag cell neurons in intact abdominal ganglia.A. Left, Extracellular recordings showing the onset of afterdischarge in response to stimulation of the pleuroabdominal connective nerve (20 V, 2.5 msec, 6 Hz). Subsequent stimulus trains fail to evoke afterdischarge (middle traces). Application of 10 μm paxilline restores spontaneous firing (bottom trace). Right, Histograms showing the mean duration of discharge in previously unstimulated clusters, refractory clusters, and refractory clusters treated with 10 μm paxilline (n = 6).B, The BK activator phloretin produces a refractory-like state in clusters of bag cell neurons. Left, Extracellular recordings showing the lack of response of previously unstimulated bag cell clusters exposed to phloretin (100 μm). When phloretin was washed out of the recording chamber, partial discharges were recovered (bottom trace). Right, Histograms showing the mean duration of discharge in previously unstimulated clusters exposed to 100 μm phloretin and after washout of phloretin (n = 5).