Apamin-sensitive small conductance calcium-activated potassium channels, through their selective coupling to voltage-gated calcium channels, are critical determinants of the precision, pace, and pattern of action potential generation in rat subthalamic nucleus neurons in vitro

Abstract

Distinct activity patterns in subthalamic nucleus (STN) neurons are observed during normal voluntary movement and abnormal movement in Parkinson's disease (PD). To determine how such patterns of activity are regulated by small conductance potassium (SK)/calcium-activated potassium (KCa) channels and voltage-gated calcium (Cav) channels, STN neurons were recorded in the perforated patch configuration in slices, [which were prepared from postnatal day 16 (P16)-P30 rats and held at 37 degrees C] and then treated with the SK KCa channel antagonist apamin or the SK KCa channel agonist 1-ethyl-2-benzimidazolinone or the Cav channel antagonists w-omega-conotoxin GVIA (Cav2.2-selective) or nifedipine (Cav1.2-1.3-selective) [corrected]. In other experiments, fura-2 was introduced as an indicator of intracellular calcium dynamics. A component of the current underlying single-spike afterhyperpolarization was sensitive to apamin, phase-locked to calcium entry via Cav2.2 channels, and necessary for precise, autonomous, single-spike oscillation. SK KCa/Cav2.2 channel coupling did not underlie spike-frequency adaptation but limited activity in response to current injection by encoding the accumulation of intracellular calcium, maintained the characteristic sigmoidal frequency-intensity relationship and generated a post-train afterhyperpolarization. In addition, SK KCa channels terminated rebound burst activity more effectively in neurons with short-duration bursts (<100 msec) than neurons with long-duration bursts (>100 msec), presumably through their activation by Cav3 channels. Cav1.2-1.3 channels were not strongly coupled to SK KCa channels and therefore supported secondary range and long-duration rebound burst firing. In summary, SK KCa channels play a fundamental role in autonomous, driven, and rebound activity and oppose the transition from autonomous, rhythmic, single-spike activity to burst firing in STN neurons.

Figure 1.

A major component of single-spike afterhyperpolarization is mediated by SK K_Ca channels. A, B, In voltage-clamp, a single unclamped action potential was induced by stepping from a holding potential of -65 to 20 mV for 10 msec. The membrane potential was then returned to -65 mV, and the current underlying afterhyperpolarization was studied. The single spike-evoked current was sensitive to apamin. B, Under current-clamp, the reduction in apamin-sensitive current manifested itself as a reduction in single-spike afterhyperpolarization and as a general depolarization (same cell as in A). Note also the steepening of the voltage trajectory in the interspike interval and the rise in the threshold for action potential generation. C, Application of the selective SK channel agonist EBIO markedly increased the magnitude of current that was evoked by the protocol described in A. D, Current-clamp recording of the same neuron in C. EBIO increased the magnitude and duration of single-spike afterhyperpolarization, which led a reduction in the frequency of autonomous oscillation.

Figure 2.

SK K_Cachannels are critical for the rhythmicity of autonomous activity. A-C, Activity of a representative neuron under control conditions and after treatment with apamin. Interspike interval histograms generated from 100 interspike intervals of the activity associated with these conditions are shown to the right. A, Precise single-spike firing was observed before drug application (CV = 0.05; frequency = 21.8 Hz). B, C, Partial inhibition (B) and complete inhibition (C) of the current with 500 pm apamin and 100 nm apamin, respectively, reduced the precision and increased the frequency of activity (500 pm apamin: CV = 0.1, frequency = 28.1 Hz; 100 nm apamin: CV = 0.18, frequency = 27.5 Hz). D, Population CV data. Nonparametric paired comparisons revealed that the mean CV in control was not significantly different from the mean CV in 10 pm apamin or the mean CV in 100 pm apamin. The mean CV in control was however significantly different from the mean CV in concentrations of apamin ≥500 pm. Calibration in C also applies to A and B.

Figure 3.

Calcium dynamics faithfully track the autonomous generation of action potentials. A, B, Simultaneous electrical (A) and fluorescent (B) recordings from the soma of a spontaneously active STN neuron. Oscillations in membrane potential are phase-locked to oscillations in intracellular calcium levels. C, Using the superimposition of multiple cycles of autonomous activity and related calcium dynamics, more precise temporal resolution of calcium dynamics was achieved (see Results). Calcium levels fell to baseline levels immediately before the generation of an action potential, rose during and after the generation of an action potential, and reached their maximum during the single-spike afterhyperpolarization. Calibration in B also applies to A.

Figure 4.

SK K_Ca channels influence the sensitivity of firing to depolarizing input. A-D, Driven firing was augmented in a dose-dependent manner by the application of apamin. Apamin increased the frequency of firing in response to current injection in both the primary and secondary firing ranges. The f-I relationship was shifted leftwards (Bi, C) and the gradient of the primary range (B, D) resembled that of the secondary range. Thus, apamin at concentrations >500 and 100 pm significantly decreased the current required for half-maximal firing (C) and increased the gradient of the primary range (D), respectively. E, F, Driven firing was markedly reduced by the application of EBIO. Fi, The f-I relationship of the neuron in E was shifted right by EBIO. Fii, The gradients of primary and secondary range firing were reduced by SK K_Ca channel activation, but the ratio of the sensitivities of primary to secondary range firing were unaltered.

Figure 5.

Intratrain spike-frequency dynamics are not controlled by SK K_Ca channels. A-D, Comparison of firing of similar frequency in control conditions and in the presence of apamin revealed that the pattern of spiking within a driven train was not altered by apamin. The speed-up in firing, which was followed by minor spike-frequency adaptation at high frequencies of activity was present in control conditions and in the presence of apamin. Calibration in C also applies to A and B.

Figure 6.

SK K_Ca channels in part underlie post-train afterhyperpolarization. A-I, High-frequency firing led to the accumulation of post-train apamin-sensitive afterhyperpolarization, which delayed the resumption of spontaneous activity. A-F, Post-train afterhyperpolarization was reliably reduced in duration and magnitude by the application of apamin. The abolition of apamin-sensitive post-train afterhyperpolarization either increased (A-C) or reduced (C-E) the time for the resumption of spontaneous activity. Thus, in the absence of SK K_Ca channel activation, the manner in which spontaneous activity resumes after high-frequency activity is more heterogeneous. G-I, EBIO consistently increased the magnitude and duration of post-train afterhyperpolarization and the time for the resumption of spontaneous activity. Calibration in A also applies to B. Calibration in D also applies to E. Calibration in G also applies to H.

Figure 7.

SK K_Ca channels underlie the build-up of post-train afterhyperpolarization but do not underlie the reduction in intratrain spike frequency during successive cycles of driven activity. A-E, An STN neuron was driven with repeated cycles of current injection: 150 pA for 100 msec, which was repeated five times with an interval of 100 msec. The post-train afterhyperpolarization, which increased during successive cycles of activity (A) was apamin-sensitive (B). The successive decline in intratrain firing was, however, apamin-insensitive and was associated with the marked accommodation and broadening of action potentials (A-E). Calibration in A also applies to B. Calibration in C also applies to D.

Figure 8.

Intracellular calcium levels accumulate during driven repetitive activity. Combined electrical and fluorescent measurements indicated that intracellular calcium accumulated when repetitive firing was driven at frequencies of >10 Hz. Intracellular calcium did not return to baseline levels during repetitive firing but did return slowly to baseline levels after driven firing over a period of several seconds. There was a linear relationship between the number of action potentials generated by current injection and the peak level of intracellular calcium for firing frequencies >10 Hz.

Figure 9.

SK K_Ca channels differentially sculpt rebound activity in neurons with short- and long-duration rebound bursts. A, The application of 100 nm apamin to a neuron with a rebound burst response of <100 msec extended greatly the duration and intensity of rebound activity at all levels of preceding hyperpolarization. B, In contrast, more modest effects on rebound activity were observed when 100 nm apamin was applied to a neuron with a long-duration rebound burst response (> 500 msec) in control conditions. C, Application of EBIO consistently shortened the duration of rebound bursts evoked after hyperpolarization to a range of membrane potentials. Calibration in C also applies to A and B.

Figure 10.

Action potential-independent calcium entry during rebound activity. A, B, Combined fluorescent and electrical recordings subthreshold and suprathreshold rebound responses in an STN neuron (inset). A, After the termination of a hyperpolarizing current step, a low-threshold calcium spike was generated without accompanying action potentials. The calcium spike was associated with a marked increase in intracellular calcium in both the soma (black trace) and dendrites (gray trace). B, In the same neuron, the early phase of a suprathreshold rebound response was followed by a later and longer lasting subthreshold response that was also associated with an increase in calcium in the soma and dendrites of the STN neuron. Calibration in B also applies to A.

Figure 11.

SK K_Ca channel blockade and hyperpolarization transform rhythmic single-spike activity into rhythmic burst activity. A, B, Tonic, slow, rhythmic single-spike activity (A) was transformed into tonic, rhythmic burst activity when SK K_Ca channels were blocked (B). C, D, Note also that after SK K_Ca channel blockade, the predominant frequency in the Lomb periodogram switched from a frequency that was similar to the mean frequency of single-spike activity (C) to a frequency that was similar to the frequency at which bursts occurred (D). The horizontal line in the Lomb periodograms denotes a level of significance of p = 0.05. Calibration in A also applies to B.

Figure 12.

SK K_Ca channels are strongly coupled to Ca_v2.2 channels. A, B, Rhythmic autonomous oscillation and single-spike afterhyperpolarization were disrupted by the application of ω-conotoxin GVIA in a manner that was similar to the effects of apamin. C, D, Ca_v2.2 channel blockade also increased the frequency of firing in response to current injection, except for the highest frequencies of activity. The gradient of the primary range of the f-I relationship was increased (Di, Dii). Although secondary range firing was generally enhanced by Ca_v 2.2 channel blockade, the gradient of the secondary range of the f-I relationship was decreased (Di, Dii). E, F, In clear contrast to the effects of SK K_Ca channel blockade, the duration of rebound activity was not altered by Ca_v2.2 channel blockade. Calibration in B also applies to A.

Figure 13.

Ca_v1.2-1.3 channels are weakly coupled to SK K_Ca channels and contribute to secondary range and long-duration rebound burst firing. A, B, Blockade of Ca_v1.2-1.3 channels produced a slight disruption in the precision of autonomous single-spike activity without a clear effect on single-spike afterhyperpolarization. C, D, Blockade of Ca_v1.2-1.3 channels had little effect on primary range firing but clearly reduced the frequency of secondary range firing and the gradient of the secondary range of the f-I relationship. E-H, Blockade of Ca_v1.2-1.3 channels had no effect on short-duration rebound activity (E, F) but significantly reduced the duration of long-duration rebound activity (G, H). Calibration in B also applies to A.