Elsevier

Neuropharmacology

Volume 60, Issue 4, March 2011, Pages 653-661
Neuropharmacology

Involvement of inward rectifier and M-type currents in carbachol-induced epileptiform synchronization

https://doi.org/10.1016/j.neuropharm.2010.11.023Get rights and content

Abstract

Exposure to cholinergic agonists is a widely used paradigm to induce epileptogenesis in vivo and synchronous activity in brain slices maintained in vitro. However, the mechanisms underlying these effects remain unclear. Here, we used field potential recordings from the lateral entorhinal cortex in horizontal rat brain slices to explore whether two different K+ currents regulated by muscarinic receptor activation, the inward rectifier (KIR) and the M-type (KM) currents, have a role in carbachol (CCh)-induced field activity, a prototypical model of cholinergic-dependent epileptiform synchronization. To establish whether KIR or KM blockade could replicate CCh effects, we exposed slices to blockers of these currents in the absence of CCh. KIR channel blockade with micromolar Ba2+ concentrations induced interictal-like events with duration and frequency that were lower than those observed with CCh; by contrast, the KM blocker linopirdine was ineffective. Pre-treatment with Ba2+ or linopirdine increased the duration of epileptiform discharges induced by subsequent application of CCh. Baclofen, a GABAB receptor agonist that activates KIR, abolished CCh-induced field oscillations, an effect that was abrogated by the GABAB receptor antagonist CGP 55845, and prevented by Ba2+. Finally, when applied after CCh, the KM activators flupirtine and retigabine shifted leftward the cumulative distribution of CCh-induced event duration; this effect was opposite to what seen during linopirdine application under similar experimental conditions. Overall, our findings suggest that KIR rather than KM plays a major regulatory role in controlling CCh-induced epileptiform synchronization.

Research highlights

► In rat EC, KIR blockade induced field discharges and enhanced CCh-induced epileptiform activity. ► KIR activation suppressed CCh-induced field discharges. ► KM inhibition did not induce field discharges but enhanced CCh-induced epileptiform activity. ► KM activation abolished CCh-induced ictal- but not interictal-like events.

Introduction

Cholinergic agonists like carbachol (CCh) or pilocarpine, by activating M1 muscarinic receptors (Cruickshank et al., 1994, Bymaster et al., 2003), induce seizure activity when administered in vivo (Turski et al., 1983), and epileptiform discharges in brain slices (Dickson and Alonso, 1997). It is still unclear which of the different transductional events activated upon M1 receptor stimulation is responsible for the appearance of this synchronous activity. A wealth of experimental evidence points to the activation of non-specific cation currents as the major factor involved even though the identity of these currents remains poorly defined. D’Antuono et al. (2001) provided evidence for a major role of the so called ICAN current (Colino and Halliwell, 1993), a non-specific cationic current activated by [Ca2+]i increases, which induces the appearance of depolarizing plateau potentials in limbic areas involved in temporal lobe epilepsy (TLE) (see Gloor, 1997) including the subiculum (D’Antuono et al., 2001), the entorhinal cortex (EC) (Klink and Alonso, 1997), and the hippocampal CA1 subfield (Fraser and MacVicar, 1996). By contrast, Egorov et al. (2003) provided evidence for a CCh-activated Ca2+-independent non-specific cation channel.

In addition to non-specific cationic channels, CCh interferes with a number of additional targets, which may play a relevant role in CCh-induced epileptiform discharges. In particular, M1 receptors activate phospholipase C (PLC), which, in turn, leads to a depletion of phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid crucially involved in the control of a wide variety of ionic currents (Suh and Hille, 2005). Among PIP2-modulated currents are inward rectifiers (KIR) (Huang et al., 1998, Sohn et al., 2007, Carr and Surmeier, 2007) and the M-current (KM), which may therefore regulate the genesis or maintenance of CCh-induced epileptiform discharges. KIR-M1receptor interaction may be particularly relevant to TLE, since inward rectifier K+ channels are highly expressed in the hippocampus and in the EC (Karschin et al., 1996), where they participate in TLE pathogenesis (Young et al., 2009). Depending upon their subunit composition, KIR may be open at resting conditions (constitutively active KIR) and control resting membrane potential, or require the stimulation of G-protein-coupled receptors to become active (Nichols and Lopatin, 1997). The latter, also referred to as GIRK channels, largely mediate the responses to inhibitory neurotransmitters (Mark and Herlitze, 2000).

KM, as well, is highly expressed in the hippocampus (Cooper et al., 2001), where it controls neuronal excitability by regulating action potential frequency adaptation (Yue and Yaari, 2004), after-depolarization/after-hyperpolarization amplitude and duration (Yue and Yaari, 2004) and, in some instances, resting membrane potential (Shah et al., 2008). Moreover, KM modulates intrinsic firing properties and subthreshold membrane oscillations in EC cells (Yoshida and Alonso, 2007), and the epileptiform activity induced by perfusion with low Mg2+-containing medium in the hippocampus (Qiu et al., 2007).

Despite these evidences, the possible involvement of KIR and KM in epileptiform synchronization consequent to cholinergic activation remains unexplored. Therefore, we employed field potential recordings from the EC, a limbic area having a major role in TLE (Du et al., 1995, de Guzman et al., 2008) and in generating the epileptiform activity induced by muscarinic agonists in brain slices (Nagao et al., 1996), along with pharmacological manipulations aimed at modulating KIR and KM, to investigate the roles of these two K+ currents in CCh-elicited synchronous activity. Our data suggest that KIR exerts a major regulation of CCh-induced epileptiform synchronization, whereas KM appears to play a complementary modulatory role.

Section snippets

Methods

Brain slices were obtained from adult male Sprague–Dawley rats (Charles River, St. Constant, QC, Canada), 2–3 months of age (250–400 g). Animal housing and experimental procedures were performed according to the recommendations of the Canadian Council on Animal Care following a research protocol approved by the McGill Animal Care Committee. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Combined hippocampus-EC slices were obtained as described by de

CCh-induced epileptiform discharges in the EC

Bath-application of CCh (100 μM) readily (10–30 min) induced the generation of synchronized oscillatory field potentials in the lateral EC in all slices (n = 32; Fig. 1A). Two types of epileptiform activity could be distinguished according to their duration (cf. de Guzman et al., 2004): (i) events shorter than 4 s (Fig. 1A, arrows), thereafter defined as ‘interictal-like’ and (ii) events longer than 4 s, termed as ‘ictal-like’ (Fig. 1A, dashed line). As indicated by the frequency distribution

Discussion

The present study investigated whether KIR or KM, two K+ currents that are influenced by muscarinic receptor activation, have a role in generating and/or maintaining CCh-induced synchronous activity in the rat lateral EC in an in vitro brain slice preparation. By using field potential recordings, we found that CCh-induced epileptiform discharges are facilitated by KIR blockade and suppressed by KIR activation; in addition, they are tuned by KM but cannot be reproduced by the pharmacological

Conclusive remarks

We have shown in the present study that two different K+ currents, both sensitive to muscarinic activation, exert distinct modulatory effects on CCh-induced epileptiform activity. Specifically, while KM appears to play only a limited role, KIR seems to be a major regulator of CCh-induced events. Further studies are needed to investigate whether these currents might be useful pharmacological targets to counteract muscarinic-dependent mechanisms possibly involved in TLE (Friedman et al., 2007).

Acknowledgements

This study was supported by the following grants: Canadian Institutes of Health Research (Grant MOP-8109) and the Savoy Foundation to, M.A., E-RARE and Telethon GGPO 7125 to M.T., PRIN 2007 to M.C.; GP received fellowships from Epilepsy Canada and the Savoy Foundation.

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