Caesium blocks depolarizing after-potentials and phasic firing in rat supraoptic neurones

Abstract

1. The effects of Cs+ on the action potential, post-train after-hyperpolarization (AHP), Ca2+-dependent post-spike depolarizing after-potential (DAP) and phasic firing were examined during intracellular recordings from magnocellular neurosecretory cells (MNCs) in superfused rat hypothalamic explants. 2. Extracellular Cs+ reversibly inhibited (IC50, approximately 1 mM) DAPs, and associated after-discharges, that followed brief spike trains in each of sixteen cells tested. Although bath application of Cs+ also provoked a small reversible depolarization, inhibition of the DAP was retained when membrane voltage was kept constant by current injection. 3. Application of Cs+ had no significant effects on spike duration (n = 8), frequency-dependent spike broadening (n = 8), spike hyperpolarizing after-potentials (n = 14), or the amplitude of the isolated AHP (n = 7). Caesium-evoked inhibition of the DAP, therefore, does not result from diminished spike-evoked Ca2+ influx, and may reflect direct blockade of the conductance underlying the DAP. 4. Inhibition of the DAP was associated with an enhancement of the amplitude and duration of the AHP, indicating that the currents underlying the AHP and the DAP overlap in time following a train of action potentials, and that the relative magnitude of these currents is an important factor in determining the shape and time course of post-train after-potentials. 5. Bath application of Cs+ reversibly abolished phasic firing in each of seven cells tested. This effect was reversible and persisted at all subthreshold voltages tested. These results indicate that the current underlying the DAP is necessary for the genesis of plateau potentials and phasic firing in MNCs.

Figure 1. Effects of extracellular Cs⁺ on post-train DAPs

A, chart recording from a supraoptic neurone impaled in a superfused hypothalamic explant. Individual vertical deflections are spike trains (full amplitude not shown) comprising 3 action potentials each, evoked by a brief current pulse (0.2 nA, 50 ms). Each train is followed by a slowly decaying DAP. Note that consecutive application of 2 mM (lower bar) and 4 mM (upper bar) Cs⁺ provokes an increasing depolarization (dashed line; −59 mV) associated with progressive inhibition of DAPs. The traces in B are excerpts of the recording shown in A (lettered arrows) showing the DAPs recorded before (a), in the presence of 4 mM Cs⁺ (b), and following washout (c).

Figure 2. Cs⁺-mediated inhibition of the DAP is not voltage dependent

A, chart recording of membrane voltage (upper trace) showing the effects of 3 mM Cs⁺ (bar) on DAPs following trains of 4 action potentials triggered by depolarizing current pulses (upward deflections in lower trace). In this experiment a variable amount of inward current (lower trace) was injected into the cell to maintain a constant initial potential (−57 mV) during exposure to Cs⁺. The traces in B are excerpts of the recording shown in A. Note that compared with control (a), addition of Cs⁺ first inhibited the after-discharge (b) and then caused a further decrease in the amplitude of the DAP (c). The effects were reversible upon washout of Cs⁺ (d). Full spike amplitude not shown.

Figure 3. Inhibition of DAPs by Cs⁺ is dose dependent

A, excerpts of an intracellular recording obtained from an MNC in which DAP amplitude was examined under control conditions and in the presence of different concentrations of Cs⁺ (as indicated). The traces are aligned vertically along the time at which a train of 3 spikes (clipped amplitude) was evoked (arrow) by a current pulse to elicit a post-train DAP (dashed line, −62 mV). The full amplitude of the AHP is not shown, except for the trace recorded in 10 mM Cs⁺. B, the graph plots the relative amplitude of DAPs (% of control) recorded from MNCs during bath application of different concentrations of Cs⁺. Note that half-maximal inhibition occurred near 1 mM. Open symbols plot data obtained from single cells whereas filled symbols show means ± s.e.m. The number of cells from which the mean was calculated is shown in parentheses beside each point.

Figure 4. Effects of Cs⁺ on other Ca²⁺-dependent parameters

Each panel shows voltage traces recorded in the presence and absence of 3 mM Cs⁺ (all are superimposed except in B where only the spikes evoked by a single train are superimposed) to illustrate effects on: A, action potential (AP) duration measured at half-amplitude (e.g. horizontal arrow), relative to baseline (dashed line); B, AP broadening, measured as the percentage change in duration observed between the first and the last AP evoked by a train; C, HAP amplitude (e.g. vertical arrow); and D, AHP amplitude relative to baseline. All action potentials (A and C) were evoked from a constant initial membrane potential and a silent interval of at least 20 s occurred before each test. The spike trains in B and D (clipped amplitude) were evoked by a depolarizing current pulse applied every 20 s and the initial membrane potential was maintained constant by DC current injection. The right panels are bar histograms showing the effects of 3-6 mM Cs⁺ on mean (+ s.e.m.) AP duration (A; % of control; n = 8), AP broadening (B; % broadening; n = 8) HAP amplitude (C; % of control; n = 14) and AHP amplitude (D; % of control; n = 7). Note that none of the parameters were significantly affected (i.e. P > 0.05; see text for absolute values).

Figure 5. Blockade of the DAP enhances the overlapping AHP

A, superimposed traces showing post-train (arrow; spike amplitude not shown) after-potentials comprising AHP (below the dashed line) and DAP (above the dashed line) components, recorded in the absence (control) and presence (Cs⁺) of 3 mM extracellular Cs⁺. B, bar histograms illustrating mean (+ s.e.m.; n = 11) relative changes in DAP amplitude, AHP amplitude and AHP duration, during application of 3-6 mM Cs⁺. □, control; ▪, Cs⁺. Note that * indicates P < 0.05 (see text for absolute values).

Figure 6. Effects of Cs⁺ on phasic firing

Traces shown are excerpts of an intracellular voltage recording from a supraoptic MNC obtained under control conditions (top), in the presence of 5 mM Cs⁺ (3 middle panels), and following return to ACSF (wash; bottom). The values shown above and to the left of each trace indicate the time at which the corresponding excerpts begin, relative to the onset of the application of Cs⁺. The values above and to the right of each trace indicate the holding current, relative to control, as well as the membrane voltage observed between action potentials. This cell showed spontaneous phasic firing with no current injected under control conditions. Bath application of Cs⁺ caused a depolarization associated with the appearance of continuous firing (0 pA). Sustained hyperpolarization by DC current injection (-110 pA) reduced the rate at which the cell fired continuously, or provoked slow irregular firing (e.g. -130 pA), but could not restore phasic firing. Further hyperpolarization of this cell abolished spontaneous firing (+60 min, -270 pA, −65 mV; not shown).

Figure 7. Cs⁺ blocks DAPs following single spikes

The traces shown are brief excerpts of the recording obtained from the cell shown in Fig. 6. The left panels show examples of the initial action potentials associated with the onset of 4 spontaneous phasic bursts recorded under control conditions. The right panels show isolated spikes, and spike doublets, recorded from the same cell while phasic activity was abolished by the application of 5 mM Cs⁺. Note that the loss of phasic firing is associated with the absence of DAPs following single action potentials, and of their summation during consecutive spikes.