Protein kinase C-dependent modulation of Na+ currents increases the excitability of rat neocortical pyramidal neurones

Abstract

The effect of the protein kinase C (PKC) activator 1-oleoyl-2-acetyl-sn-glycerol (OAG) on TTX-sensitive Na+ currents in neocortical pyramidal neurones was evaluated using voltage-clamp and intracellular current-clamp recordings. In pyramid-shaped dissociated neurones, the addition of OAG to the superfusing medium consistently led to a 30% reduction in the maximal peak amplitude of the transient sodium current (I(Na,T)) evoked from a holding potential of -70 mV. We attributed this inhibitory effect to a significant negative shift of the voltage dependence of steady-state channel inactivation (of approximately 14 mV). The inhibitory effect was completely prevented by hyperpolarising prepulses to potentials that were more negative than -80 mV. A small but significant leftward shift of INa,T activation was also observed, resulting in a slight increase of the currents evoked by test pulses at potentials more negative then -35 mV. In the presence of OAG, the activation of the persistent fraction of the Na+ current (INa,P) evoked by means of slow ramp depolarisations was consistently shifted in the negative direction by 3.9+/-0.5 mV, while the peak amplitude of the current was unaffected. In slice experiments, the OAG perfusion enhanced a subthreshold depolarising rectification affecting the membrane response to the injection of positive current pulses, and thus led the neurones to fire in response to significantly lower depolarising stimuli than those needed under control conditions. This effect was attributed to an OAG-induced enhancement of INa,P, since it was observed in the same range of potentials over which I(Na,P) activates and was completely abolished by TTX. The qualitative firing characteristics of both the intrinsically bursting and regular spiking neurones were unaffected when OAG was added to the physiological perfusing medium, but their firing frequency increased in response to slight suprathreshold depolarisations. The obtained results suggest that physiopathological events working through PKC activation can increase neuronal excitability by directly amplifying the I(Na,P)-dependent subthreshold depolarisation, and that this facilitating effect may override the expected reduction in neuronal excitability deriving from OAG-induced inhibition of the maximal INa, T peak amplitude.

Figure 1. Effect of 2 μm OAG on I_Na,T steady-state inactivation

Aa-c, current traces obtained in the same neurone by means of a depolarising step to −15 mV preceded by a 300 ms prepulse at various potentials (the stimulus protocol is shown in the inset of B). The largest current peaks were obtained after a prepulse to −100 mV and were unchanged after OAG, but the inhibition of the current peak becomes evident after prepulses to −70 mV and −60 mV. B, steady-state inactivation curve obtained by plotting the current peaks (normalised to their maximal values) versus the prepulse potential. The continuous lines are fitted curves obtained by means of a Boltzmann function applied to the data points calculated under control conditions and in the presence of OAG.

Figure 2. Effects of 2 μm OAG on I_Na,T activation and fast inactivation time course

Aa and b, I_Na,T traces recorded in a representative neurone in response to two different voltage steps under control conditions and in the presence of OAG. Ba and b, I_Na,T traces evoked in a neurone held at −90 mV, showing a slight increase of the current evoked using a depolarising pulse to −45 mV, but no decrease in maximal current peak amplitude in response to a depolarising pulse to −15 mV. C, voltage-dependent Na⁺ conductance evaluated in 14 neurones (the stimulus protocol is shown in the inset); the curves give the Boltzmann function fit applied to the data points calculated under control conditions and in the presence of OAG, normalised to the maximal values, respectively, obtained in the two experimental conditions. D, plot of the voltage dependence of the fast time constants (τ_i) of the I_Na,T decay (symbols as in C). In the inset Ac, the same traces as in Ab are scaled at the same amplitude to show the overlap of the fast inactivation time course under control conditions and in the presence of OAG.

Figure 3. Partial recovery of I_Na,T peak amplitude obtained after OAG-induced inhibition

A, I_Na,T traces using a step potential to −15 mV under control conditions, in the presence of OAG and 10 min after the onset of perfusion with 2 μm OAG plus the PKC inhibitor H7 (10 μm). B, time course of the reduction and recovery of the maximal I_Na,T peak amplitude, respectively, during OAG perfusion and in the presence of OAG plus H7. C, current-voltage relationship obtained under the three different conditions. D, mean decrease and subsequent recovery of the I_Na,T peak amplitude (step potential to −15 mV) in four neurones perfused with OAG and with OAG plus H7.

Figure 4. Effect of 2 μm OAG on I_Na,P

A, I_Na,P evoked by a slow ramp voltage protocol (top trace). B, I_Na,P traces obtained under control conditions and in the presence of OAG added to the PKC inhibitor H7 (10 μm). C, I_Na,P traces recorded at the onset and 10 min after the onset of perfusion with DMSO at the same concentration used to dissolve OAG. D, mean of I_Na,P traces obtained from 14 neurones; the mean traces obtained in five of these neurones after TTX perfusion is also shown. E, plot of I_Na,P activation curve: the data points are the mean conductances normalised to maximal values under control conditions and in the presence of OAG (n= 14) plotted against the command potentials. The continuous line is the mean of 14 fitting curves obtained by means of a Boltzmann relationship.

Figure 5. Effect of OAG on I_Na,P evoked by voltage steps

A, plot of the Na⁺ conductance calculated from the I_Na,P amplitude at the end of 350 ms voltage pulses (the stimulus protocol is shown in the inset). The data points are the mean conductance values obtained by measuring (n= 6 neurones) the current plateau under control conditions and in the presence of OAG, plotted against the command potentials and fitted by means of a Boltzmann function. The TTX-subtracted current traces recorded in a representative neurone in response to a voltage step at −35 mV are shown in B.

Figure 6. Effect of 12.5 μm OAG on subthreshold and threshold membrane behaviour

Two representative neurones perfused with modified ACSF containing 40 mm TEA, 1 mm BaCl₂, 10 mm CsCl and 2 mm CoCl₂. A, in a potential range close to the firing threshold, the membrane deflection in response to depolarising current pulses is greater in the presence of OAG than under control conditions, and the current pulse leading to AP generation is significantly smaller. The addition of 1 μm TTX leads to merely passive membrane behaviour. B, voltage-current plot showing the rectifying time course of membrane deflection in response to a family of depolarising pulses, which is enhanced in the presence of OAG and abolished by TTX. C, superimposition of two sweeps selected from the recordings obtained in the same neurone as in A, showing the complete block of depolarising rectification after the addition of TTX. D, preincubation with the PKC inhibitor chelerythrine (20 μm) prevents the effect of OAG. The calibration values apply to all panels.

Figure 7. OAG effect on an IB neurone recorded in physiological ACSF

A, the injection of a low amplitude depolarising current pulse equal to that sustaining a subthreshold membrane depolarisation under control conditions (left panel, arrow) is capable of leading the neurone to fire with a burst in the presence of OAG (12.5 μm; right panel). B, threshold depolarisation under control conditions (dashed line) needs a larger pulse than that necessary in the presence of OAG.

Figure 8. Effects of OAG on the firing characteristics of two different neurones

The left panels show neuronal firing under control conditions; the right panels show the response to an identical current pulse injection in the presence of OAG (12.5 μm). A, threshold response of an IB neurone to a depolarising pulse injection (a); in the presence of OAG, an equal pulse led to a discharge with repetitive bursts (b). Under control conditions, the same neurone responding to a larger current injection with repetitive short burst (c) changes its firing behaviour to two initial bursts followed by a tonic rhythmic discharge of individual APs superimposed on a membrane depolarisation (d). B, threshold (a) and slightly suprathreshold (c) firing of an RS neurone showing an increased firing rate in the presence of OAG (b and d).

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