Distinct contributions of high- and low-voltage-activated calcium currents to afterhyperpolarizations in cholinergic nucleus basalis neurons of the guinea pig

Abstract

The contributions made by low- (LVA) and high-voltage-activated (HVA) calcium currents to afterhyperpolarizations (AHPs) of nucleus basalis (NB) cholinergic neurons were investigated in dissociated cells. Neurons with somata >25 microM were studied because 80% of them stained positively for choline acetyltransferase and had electrophysiological characteristics identical to those of cholinergic NB neurons previously recorded in basal forebrain slices. Calcium currents of cholinergic NB neurons first were dissected pharmacologically into an amiloride-sensitive LVA and at least five subtypes of HVA currents. Approximately 17% of the total HVA current was sensitive to nifedipine (3 microM), 35% to omega-conotoxin-GVIA (200-400 nM), 10% to omega-Agatoxin-IVA (100 nM), and 20% to omega-Agatoxin-IVA (300-500 nM), suggesting the presence of L-, N-, P-, and Q-type channels, respectively. A remaining current (R-type) resistant to these antagonists was blocked by cadmium (100-200 microM). We then assessed pharmacologically the role that LVA and HVA currents had in activating the apamin-insensitive AHP elicited by a long train of action potentials (sAHP) and the AHP evoked either by a short burst of action potentials or by a single action potential (mAHP) that is known to be apamin-sensitive. During sAHPs, approximately 60% of the hyperpolarization was activated by calcium flowing through N-type channels and approximately 20% through P-type channels, whereas T-, L-, and Q-type channels were not involved significantly. In contrast, during mAHPs, N- and T-type channels played key roles (approximately 60 and 30%, respectively), whereas L-, P-, and Q-type channels were not implicated significantly. It is concluded that in cholinergic NB neurons various subtypes of calcium channels can differentially activate the apamin-sensitive mAHP and the apamin-insensitive sAHP.

Fig. 1.

Large dissociated NB neurons are cholinergic.A, Histogram of ChAT-positive and ChAT-negative neurons showing that cells >25 μm are mostly cholinergic (>80%). B, In a characteristic cholinergic NB neuron, a hyperpolarizing current step typically elicited only a weak inward rectifying current (I_h;asterisk), which was followed by an A-type rectification (arrowhead) and burst firing. C, At a membrane potential of −62 mV, the same cell responded to a depolarizing current step with regular firing at a low frequency and displayed an AHP at the end of the step. D, In most of the cells, application of a depolarizing step from a negative membrane potential (−80 mV) elicited repetitive bursts of action potentials (enlarged in the inset) that usually were preceded by a delay in firing, presumably because of the A-type current (arrowhead).

Fig. 2.

Pharmacological characterization of calcium currents. A, Plot of the peak calcium current versus time during cumulative applications of 3 μm nifedipine, 400 nm cono-GVIA, 100 nm Aga-IV, 400 nm Aga-IVA, and 200 μm cadmium.B₁, Whole-cell leak-subtracted current traces (same experiment as in A) elicited by a 40 msec depolarizing step to 0 mV from a V_h of −90 mV. In this cell 12% of the total HVA calcium current was blocked by nifedipine, 37% by cono-GVIA, 7% by Aga-IVA (100 nm), and 12% by Aga-IVA (400 nm), whereas the remaining current (33%) was eliminated by cadmium (200 μm).B₂, Whole-cell leak-subtracted current traces obtained with a depolarizing test pulse to −40 mV from a membrane potential of −90 mV (to activate the T-type current) showing the reversible block produced by amiloride (300 μm).C, Summary of experiments performed with calcium current antagonists, expressed in percentage of reduction over the control, for cumulative (C₁) and separate (C₂) applications. D, Peak calcium current as a function of test potential in control conditions and during cumulative applications of calcium antagonists. A LVA current was evident at potentials greater than −60 mV and was followed by currents that peaked at 0 mV.

Fig. 3.

N- and P-type calcium channels are involved in the sAHP. A, An AHP evoked by a 400 msec train of 18 depolarizations (1 msec each in duration) was greatly reduced (measured at arrow indicating the peak amplitude) by the application of 300 nm cono-GVIA. B, In another cell the application of Aga-IVA (100 nm) produced a reduction of 20% of the sAHP. C, The T-type calcium current blocker amiloride (300 μm) did not affect the sAHP. D, Histogram summarizing the percentage of reduction of the sAHP measured at the peak for separate drug applications. Spike height was truncated in A–C.

Fig. 4.

N- and T-type calcium channels are involved in the mAHP. A, An mAHP (peak amplitude indicated byarrow) was elicited by a burst of three APs from a membrane potential of −80 mV. Application of 300 nmcono-GVIA reduced the AHP by ∼64%. Note in inset that the mAHP was elicited by the same number of APs in control and in the presence of the antagonist cono-GVIA. B, Summary of results obtained for separate applications of calcium channel antagonists (cono-GVIA, 200–400 nm; nifedipine, 3 μm). C, D, Amiloride (300 μm) reduced the amplitude of the mAHP after a burst but also reduced the number of APs in the burst to a single AP.E, In mAHP activated by a single AP (1 msec depolarizing pulse from a potential of −60 mV), 300 μm amiloride blocked both the DAP (open arrowhead) and a portion of the AHP (arrow), 3 μm nifedipine had no effect (data not shown), and 400 nm cono-GVIA (added with both amiloride and nifedipine) blocked the remaining mAHP (cadmium did not reduce the AHP further). F, Summary of experiments on mAHP elicited after a single AP. Both amiloride and cono-GVIA significantly reduced the mAHP, whereas 3 μm nifedipine (added after amiloride) had no effect on the mAHP.