(+)-Dehydrofukinone modulates membrane potential and delays seizure onset by GABAa receptor-mediated mechanism in mice

Highlights

• (+)-Dehydrofukinone blunted KCl-evoked depolarization and calcium influx.

• (+)-Dehydrofukinone effects on neuronal excitability are blocked by flumazenil.

• (+)-Dehydrofukinone delays pentylenetetrazole-induced seizures in mice.

• GABAa receptors play a role in the anticonvulsant effect of (+)-dehydrofukinone.

Abstract

(+)-Dehydrofukinone (DHF), isolated from Nectandra grandiflora (Lauraceae) essential oil, induces sedation and anesthesia by modulation of GABAa receptors. However, no study has addressed whether DHF modulates other cellular events involved in the control of cellular excitability, such as seizure behavior. Therefore, the aim of the present study was to investigate the effect of DHF on cellular excitability and seizure behavior in mice. For this purpose, we used isolated nerve terminals (synaptosomes) to examine the effect of DHF on the plasma membrane potential, the involvement of GABAa receptors and the downstream activation of Ca^2 + mobilization. Finally, we performed an in vivo assay in order to verify whether DHF could impact on seizures induced by pentylenetetrazole (PTZ) in mice. The results showed that DHF induced a GABA-dependent sustained hyperpolarization, sensitive to flumazenil and absent in low-[Cl⁻] medium. Additionally, (1–100 μM) DHF decreased KCl-evoked calcium mobilization over time in a concentration-dependent manner and this effect was prevented by flumazenil. DHF increased the latency to myoclonic jerks (10 mg/kg), delayed the onset of generalized tonic-clonic seizures (10, 30 and 100 mg/kg), and these effects were also blocked by the pretreatment with flumazenil. Our data indicate that DHF has anticonvulsant properties and the molecular target underlying this effect is likely to be the facilitation of GABAergic neuronal inhibition. The present study highlights the therapeutic potential of the natural compound DHF as a suppressor of neuronal excitability.

Introduction

(+)-Dehydrofukinone (DHF), also known as dihydrokaranone, is an eremophilane-type sesquiterpenoid ketone isolated from Nectandra grandiflora Ness (Lauraceae) essential oil. Also, DHF was previously detected in Senecio and Ligularia species, Arcticum lappa and Cascalia hastata (Lizarraga et al., 2013), however, the DHF enantiomeric form was not specified in those studies. Early behavioral studies have indicated that DHF has sedative and anesthetic properties mediated by GABAergic mechanisms in fish (Heinzmann et al., 2014, Garlet et al., 2016). However, no study has addressed whether DHF modulates other cellular events involved in the control of cellular excitability, such as seizure behavior.

Seizures (lapse of attention, muscle jerks or severe and prolonged convulsions) are triggered from an imbalance of neuronal excitation and inhibition, resulting in excessive and synchronized neuronal electrical discharges (Jung, 1958, Greenfield, 2013, Staley, 2015). Recurrent seizures or epileptic episodes characterizes epilepsy, which is a neurological chronic disorder of varying etiology that affects > 50 million people worldwide (WHO, 2015). Current treatments fail to control seizures in 20–30% of the patients (Löscher et al., 2013) and new approaches that may overcome this issue are a goal of current epilepsy research. Seizures occurrence could be prevented by decreasing epileptic bursting, synchronization and seizure spread. Drugs with antiseizure properties generally inhibit synaptic excitation, enhance synaptic inhibition or modulate voltage-gated ion channels (Rogawski and Löscher, 2004, Prince et al., 2009, Löscher et al., 2013, Kaminski et al., 2014).

Failure of inhibitory neurotransmission has been implicated in epileptogenesis (González et al., 2015) and γ-aminobutyric acid (GABA) type A receptors (GABAa) are believed to play an important role in this process (Zhang et al., 2007, Schipper et al., 2015). Upon binding of the neurotransmitter GABA, the GABAa receptor allows chloride and bicarbonate ions to diffuse through the plasma membrane, which results in hyperpolarization. Neuronal cells under hyperpolarized state have higher excitation thresholds and are unlikely to open voltage-gated channels, such as voltage-gated calcium channels (VGCC) (Rogawski and Löscher, 2004, Greenfield, 2013, Miceli et al., 2015, Tritsch et al., 2016). The Ca^2 + influx triggered by depolarization promotes neurotransmitter release by vesicle recruitment in the active zone (Neher and Sakaba, 2008, Kavalali, 2014). Continued elevation of intracellular calcium levels and therefore excitatory neurotransmitter release are observed in neuronal cells during seizures (Kulak et al., 2004, Steinlein, 2014). Voltage-dependent Ca^2 + influx is suppressed by postsynaptic GABAa receptor activation, which led the cell to hyperpolarization and therefore to a potential below the VGCC opening threshold (Neumaier et al., 2015). Thus, GABAergic tone enhancement is a target to intracellular calcium and cell excitability modulation and therefore, seizure control (Steinlein, 2014, Neumaier et al., 2015, Ochoa and Kilgo, 2016). Indeed, several antiseizure drugs target the GABAergic system, such as benzodiazepines, barbiturates, tiagabine and vigabatrin (Rogawski and Löscher, 2004, Kaminski et al., 2014). The anticonvulsant properties of topiramate and valproate are a combination of GABAergic boosting and VGCC blockade (Löscher, 1999, Kaminski et al., 2014, Gao and Li, 2016). Additionally, several natural compounds that modulate GABAa receptors, such as acacetin, curcumol, eudesmin, methylapigenin and hesperidin, have shown anticonvulsant activity in pre-clinical studies (Chen et al., 2016, Ding et al., 2014, Hosseinzadeh and Parvardeh, 2004, Lin et al., 2012, Liu et al., 2015, Lin et al., 2014, Marder et al., 2003). Considering that DHF putatively interacts with GABAa receptors (Garlet et al., 2016), we hypothesized that this natural compound could impact on seizures. Therefore, the aim of the present study was to investigate the effect of DHF on synaptosomal excitability and calcium influx and seizure behavior in mice. For this purpose, we used isolated nerve terminals (synaptosomes) purified from mice cortex as a model to examine the effect of DHF on the synaptosomal plasma membrane potential, the involvement of GABAa receptors and the downstream activation of synaptosomal calcium mobilization. Synaptosomes mimic the synaptic function and are applied in the study of neuronal signaling pathways (Erecińska et al., 1996, Dunkley et al., 2008, Evans, 2015). This standardized in vitro model of neuronal terminal endings is widely used to investigate the actions of drugs, including natural compounds, on membrane potential and ion channels. (Lu et al., 2010, Kammerer et al., 2011, Kuo et al., 2012, Lin et al., 2011a, Lin et al., 2011b, Lin et al., 2012, Kamat et al., 2014, Lin et al., 2014, Chang et al., 2015a, Chang et al., 2015b, Lin et al., 2015). Finally, we performed an in vivo assay in order to verify whether DHF could influence acute seizures induced by pentylenetetrazole (PTZ). This particular model was chosen because PTZ tests in rodents predict clinical activity of antiseizure drugs with GABAergic mechanisms (Löscher, 2011).