The Journal of Neurophysiology Vol. 79 No. 3 March 1998, pp. 1579-1582
Copyright ©1998 by the American Physiological Society

RAPID COMMUNICATION

Muscarinic Inhibition of Persistent Na⁺ Current in Rat Neocortical Pyramidal Neurons

Thomas Mittmann¹ and Christian Alzheimer²

¹ Department of Neurophysiology, Ruhr-University Bochum, D-44780 Bochum; and ² Department of Physiology, University of Munich, D-80336 Munich, Germany

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Mittmann, Thomas and Christian Alzheimer. Muscarinic inhibition of persistent Na⁺ current in rat neocortical pyramidal neurons. J. Neurophysiol. 79: 1579-1582, 1998. Muscarinic modulation of persistent Na⁺ current (I_NaP) was studied using whole cell recordings from acutely isolated pyramidal cells of rat neocortex. After suppression of Ca²⁺ and K⁺ currents, I_NaP was evoked by slow depolarizing voltage ramps or by long depolarizing voltage steps. The cholinergic agonist, carbachol, produced an atropine-sensitive decrease of I_NaP at all potentials. When applied at a saturating concentration (20 µM), carbachol reduced peak I_NaP by 38% on average. Carbachol did not alter the voltage dependence of I_NaP activation nor did it interfere with the slow inactivation of I_NaP. Our data indicate that I_NaP can be targeted by the rich cholinergic innervation of the neocortex. Because I_NaP is activated in the subthreshold voltage range, cholinergic inhibition of this current would be particularly suited to modulate the electrical behavior of neocortical pyramidal cells below and near firing threshold.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Activation of muscarinic receptors exerts a wide spectrum of actions in neocortical neurons, including modulation of K⁺ currents, Ca²⁺ currents, and a nonselective cation current (Haj-Dahmane and Andrade 1996; McCormick 1993). In the present study, we investigated whether muscarinic receptor stimulation also would affect the persistent Na⁺ current (I_NaP) of neocortical neurons, a functionally important ion conductance in the subthreshold voltage range (Crill 1996). It is firmly established that Na⁺ currents can be modulated by protein kinase A- and C-mediated phosphorylation of the principal () subunit of Na⁺ channel proteins (Catterall 1992). Because muscarinic receptors use protein kinase C (PKC)-mediated phosphorylation as one of their signal transduction pathways (Durieux 1996), Na⁺ channel gating represents a putative target of cholinergic modulation in rat neocortex. With whole cell recordings from acutely isolated neocortical pyramidal cells, we report here that activation of muscarinic receptors decreases persistent Na⁺ current, without changing its voltage-dependent properties or its slow inactivation kinetics.

	METHODS

Abstract Introduction Methods Results Discussion References

Details of the slicing and dissociation procedures have been described elsewhere (Alzheimer et al. 1993a). Briefly, 400-µm-thick coronal slices were taken from sensorimotor cortex of ether- anesthetized rats 13-19 days old. Before mechanic trituration, small pieces of slice tissue were incubated for 90 min at 29°C in N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES)-buffered oxygenated saline solution containing 19 U/ml papain. Whole cell currents were evoked and recorded using an Axopatch 200 amplifier in conjunction with a TL-1 interface and pClamp 6.0 software (all from Axon Instruments). Current signals were sampled at 2-5 kHz and filtered at 1 kHz (3 dB). All recordings were performed at room temperature (21-24 °C). Membrane potential was corrected for liquid junction potential. Throughout the experiments, leakage and capacitive currents were determined from hyperpolarizing voltage steps and eliminated using the built-in compensation circuits of the amplifier. Experiments were started when stable I_NaP responses were obtained, typically 5-8 min after whole cell access was established. During the initial stabilization period, neurons usually displayed a variable degree of I_NaP runup. After that period, control recordings (n =5) showed that I_NaP remained stable for the time of experimentation (10-15 min). If not stated otherwise, holding potential was 70 mV. The bath solution contained (in mM) 130 NaCl, 3 KCl, 1.6 CaCl₂, 0.4 CdCl₂, 2 MgCl₂, 25 HEPES/NaHEPES, and 10 D-glucose (pH 7.4). The pipette solution contained (in mM) 110 Cs-gluconate, 3 MgCl₂, 5 ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid, 5 HEPES, 2 Tris-ATP, 0.3 Tris-GTP, 15 phosphocreatine, and 0.1 leupeptin (pH 7.25, adjusted with NaOH). When filled with pipette solution, electrode resistances were 4-6 M in the bath and 15-25 M in the whole cell configuration before series resistance compensation (70-75%). Drugs were bath applied by means of a multiple-inlet system allowing complete bath exchange within ~15 s. Carbachol, atropine, and mecamylamine were all purchased from Sigma (Deisenhofen, Germany). Statistics are presented as means ± SE. Statistical analyses (t-test and analysis of variance) were done with the use of Graphpad Prism 2.0.

	RESULTS

Abstract Introduction Methods Results Discussion References

To isolate voltage-dependent Na⁺ currents, Ca²⁺ and K⁺ currents were blocked by external Cd²⁺ and internal Cs⁺, respectively. When applied under these conditions, long depolarizing voltage steps or slow depolarizing voltage ramps evoked I_NaP of typical voltage dependence and amplitude (cf. Alzheimer et al. 1993b). The effect of the cholinergic agonist carbachol (20-100 µM) was tested in 25 visually identified pyramidal-shaped neurons. The nicotinic receptor antagonist, mecamylamine (10 µM), was added routinely to the bathing solutions to block nicotinic responses to carbachol. In the first set of experiments, slow depolarizing voltage ramps (-70-0 mV) were applied at 15-s intervals, and peak I_NaP amplitudes were determined before, during, and after superfusion of carbachol (Fig. 1, A and B). In 5 of 25 cells, carbachol did not affect I_NaP. In the remaining cells (80%), carbachol consistently reduced I_NaP. At 20 µM, carbachol decreased peak I_NaP to 62.4 ± 2.6% (n = 17) of control. At higher concentrations (50-100 µM), carbachol did not further decrease peak I_NaP amplitude (reduction to 59.6 ± 2.3% of control, n = 5, P = 0.59). The outward-going, nonselective cation current (I_cat) appearing at potentials more positive than 30 mV under these experimental conditions (cf. Alzheimer 1994) was not affected by carbachol (100 µM) when studied in isolation (i.e., in the presence of 1 µM tetrodotoxin, n = 4, Fig. 1C). To establish the involvement of muscarinic receptors, responses to carbachol were measured in the absence and presence of the muscarinic antagonist, atropine. Peak I_NaP amplitudes obtained in carbachol solution or in carbachol/atropine solution were normalized to the peak I_NaP amplitude recorded under control conditions. Figure 2 summarizes the results from five different neurons, where carbachol alone (20 µM) reduced peak I_NaP to 65.8 ± 3.2% of control, whereas the current attained almost its maximum value when carbachol was applied with atropine (1 µM) present in the bathing solution (94.6 ± 3.1% of control, P = 0.0002).

View larger version (19K): [in this window] [in a new window]   FIG. 1. INaP evoked by slow depolarizing voltage ramps. A: time course of carbachol (20 µM) action on peak INaP amplitude (current in absolute values). B: INaP responses taken from A at the time points indicated by like-numbers. C: blockade of INaP by tetrodotoxin revealed a cationic outward current (Icat) (cf. Alzheimer 1994) developing at potentials positive to 30 mV. In contrast to INaP, the outward current was not affected by carbachol.

View larger version (46K): [in this window] [in a new window]   FIG. 2. Cholinergic inhibition of INaP is mediated by muscarinic receptors. Atropine (1 µM) abolished reduction of INaP by carbachol (20 µM).

To determine the effects of carbachol under steady-state voltage-clamp conditions, membrane potential was depolarized using square voltage commands to various test potentials. Figure 3A shows the typical biphasic current response evoked by a sustained depolarizing voltage step to 42 mV. The instantaneous surge of transient inward current was followed by a small sustained inward current, I_NaP (cf. Alzheimer et al. 1993b). Application of 20 µM carbachol produced an apparently time-independent decrease of I_NaP that was reversible after drug wash-out. Comparison of the complete I-V curves (Fig. 3B) shows that the voltage dependence of I_NaP activation was not altered by muscarinic modulation.

View larger version (19K): [in this window] [in a new window]   FIG. 3. INaP evoked by square voltage commands. A: reversible inhibition of INaP by 20 µM carbachol. Because of high gain of recording, fast Na+ current was largely truncated. For illustration, current signals were digitally refiltered at 0.2 kHz. B: I-V relationship of sustained inward current determined at the end of each voltage step before, during, and after carbachol (20 and 100 µM).

One functionally important feature of I_NaP is the slow inactivation brought about by prolonged depolarization (Fleidervish and Gutnick 1996). To examine whether muscarinic receptor activation would affect this process, activation of I_NaP was tested using depolarizing voltage ramps of variable speed (range: 53-13 mV/s). Figure 4A depicts I_NaP responses evoked by a fast and a slow ramp protocol before and during carbachol (20 µM) application. Activation of muscarinic receptors produced about equal reduction of I_NaP independent of ramp speed and corresponding I_NaP availability. Figure 4B summarizes data from seven such experiments. Each column represents the carbachol-induced reduction in relative I_NaP at the ramp speed indicated above. Because the column means were not significantly different, carbachol is unlikely to interfere with the mechanisms underlying slow inactivation of I_NaP.

View larger version (34K): [in this window] [in a new window]   FIG. 4. INaP evoked by depolarizing voltage ramps (60-0 mV) of different speed. A: individual INaP responses recorded at the ramp speed indicated above current traces before and during carbachol (20 µM) application. B: relative reduction of peak INaP amplitude by carbachol (20 µM) was independent of ramp speed.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Our data predict that I_NaP of pyramidal cells can be modulated by the dense cholinergic innervation reaching the cerebral cortex. With the cholinergic agonist carbachol in combination with a nicotinic receptor antagonist, we found that stimulation of muscarinic receptors causes an atropine-sensitive decrease of I_NaP at all potentials without changing the voltage dependence of I_NaP activation. Unlike muscarinic- or PKC-dependent modulation of fast Na⁺ current, which not only reduces current amplitude but also slows fast inactivation (Cantrell et al. 1996; Catterall 1992), carbachol did not affect the mechanism(s) responsible for slow inactivation of I_NaP. This agrees with the notion that fast and slow Na⁺ channel inactivation are separate processes that are regulated independently (Patlak 1991). In view of the small size of I_NaP, the aim of the present study was not to obtain a complete dose-response relationship for cholinergic inhibitionof I_NaP but to establish that I_NaP is subject to muscarinic modulation. Carbachol hence was applied at a saturating concentration (20 µM) that decreased I_NaP amplitude by 38% on average. Interestingly, a similar value was reported for muscarinic inhibition of fast Na⁺ current in hippocampal neurons, with a saturating concentration of carbachol (50 µM) reducing transient Na⁺ current by ~30% (Cantrell et al. 1996).

Given the almost equal sensitivity of fast and sustained Na⁺ current to cholinergic input, the ratio of persistent to fast Na⁺ current should remain largely unaffected by muscarinic modulation. In this respect, the action of carbachol differs from effects of state-dependent Na⁺ channel blockers such as the anticonvulsant phenytoin, which suppresses I_NaP more potently than fast Na⁺ current (Chao and Alzheimer 1995). Thus phenytoin and carbachol seem to employ different mechanisms of Na⁺ current modulation. Because of its higher affinity to inactivated Na⁺ channels, phenytoin preferentially diminishes the late Na⁺ channel openings (Segal and Douglas 1997) that are thought to underlie I_NaP (Alzheimer et al. 1993a), whereas muscarinic receptor stimulation appears to reduce early and late Na⁺ channel openings to an approximately equal extent.

How does the (partial) inhibition of a small current like I_NaP compare to other actions of acetylcholine in the brain? Despite the well-established and, in part, strong effects of acetylcholine on several other ion currents (see INTRODUCTION), modulation of I_NaP will prove particularly influential when we consider the behavior of neurons below firing threshold. In this voltage range, I_NaP plays an important role because other sustained ion currents are small or absent. Consequently, I_NaP has been implicated in a number of processes typical of this voltage range, such as amplification of excitatory synaptic input in different neuronal compartments (Lipowsky et al. 1996; Schwindt and Crill 1995; Stuart and Sakmann 1995), generation of membrane oscillations (Alonso and Llinas 1989), and fine-tuning of intrinsic firing patterns (for review, see Crill 1996). Modulation of I_NaP hence would represent a powerful tool to influence one or several of these cellular functions. Apart from membrane voltage, the weight assigned to muscarinic modulation of I_NaP also will depend on the interaction among Na⁺ channels, muscarinic receptors, and other ion channels targeted by cholinergic input, as determined by their spatial distribution within the different compartments of a neuron.

	ACKNOWLEDGEMENTS

  We are grateful to L. Kargl for technical assistance.

  This work was supported in part by the Bundesministerium der Verteidigung. C. Alzheimer is a Heisenberg-Fellow of the Deutsche Forschungsgemeinschaft.

	FOOTNOTES

  Address for reprint requests: C. Alzheimer, Dept. of Physiology, University of Munich, Pettenkoferstr. 12, D-80336 Munich, Germany.

  Received 20 October 1997; accepted in final form 9 December 1997.

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