Elsevier

Neuropharmacology

Volume 44, Issue 2, February 2003, Pages 234-243
Neuropharmacology

Electroencephalographic characterisation of pentylenetetrazole-induced seizures in mice lacking the α4 subunit of the neuronal nicotinic receptor

https://doi.org/10.1016/S0028-3908(02)00369-6Get rights and content

Abstract

Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE) is associated in some kindreds with mutations in the genes encoding the α4 or β2 subunits of the neuronal nicotinic acetylcholine receptor (nAChR). Functional characterisation of the described ADNFLE mutations in oocyte preparations has produced conflicting results, with some studies suggesting hypofunction but others showing increased ligand sensitivity or delayed desensitisation. Knockout mice were studied to investigate extreme hypofunction of α4 nAChRs in vivo. Mutant (Mt) and control mice underwent epidural electroencephalographic (EEG) recording for 2 h in the untreated state and for 1 h following administration of the γ-amino butyric acid (GABA) antagonist, pentylenetetrazole (PTZ, 80 mg/kg). No spontaneous seizures occurred and no EEG differences were observed between the genotypes in drug naïve mice. Following PTZ, however, Mt mice showed markedly increased mortality compared to controls (85 vs 30%, P<0.001). Mts also had a greater number of generalised clonic seizures in the first 40 min following injection. In the same period, the EEGs of Mt mice showed an excess of spikes (P=0.033), multi-spike complexes (P=0.002) and continuous fast activity (P=0.017) compared to controls. These findings demonstrate that intact α4 nAChR subunits provide significant in vivo protection against the proconvulsant effects of GABA antagonism.

Introduction

Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE) is an inherited disorder in which seizures occur during sleep. Five mutations have been identified in ADNFLE kindreds to date and all involve the α4 or β2 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) (Steinlein et al., 1995, Steinlein et al., 1997, Hirose et al., 1999, Phillips et al., 2001, De Fusco et al., 2000). Neuronal AChRs are ligand-gated ion channels that, once activated, are permeable to sodium and calcium and cause cellular depolarisation. They are pentamers variously composed of α subunits (α2–α10) and β subunits (β2–β4) (Le Novere and Changeux, 1995, Elgoyhen et al., 2001). The most common configuration contains α4 and β2 subunits, while the α7 homooligomer also plays an important role (Gotti et al., 1997).

Activation of presynaptic nAChRs is known to facilitate the release of several neurotransmitters including γ-amino butyric acid (GABA), glutamate, dopamine and acetylcholine (ACh) itself (Wonnacott, 1997). Studies of GABAergic facilitation have primarily implicated α4/β2 nAChRs (Lena and Changeux, 1997, Lu et al., 1998). Glutamate facilitation appears to involve α7 nAChRs in many brain regions (Radcliffe and Dani, 1998, MacDermott et al., 1999) but may also involve α4/β2 nAChRs (Vidal and Changeux, 1993, Gil et al., 1997, Gioanni et al., 1999). The composition of nAChRs modulating dopamine release is uncertain; contributions from α3, α4, β2 and β4 subunits have been proposed (Grady et al., 2001, Wonnacott, 1997, MacDermott et al., 1999).

Postsynaptic nAChRs have also been demonstrated in many regions and application of nicotinic agonists in vitro can elicit cellular depolarisation and action potentials in nAChR-positive neurons (Xiang et al., 1998, Porter et al., 1999, Alkondon et al., 2000). There is also in vitro evidence implicating nAChRs in fast synaptic transmission in the hippocampus (Hefft et al., 1998, Frazier et al., 1998). Because the role of nAChRs in normal neurotransmission in vivo remains uncertain, however, aetiological hypotheses about ADNFLE have concentrated on the neuromodulatory role of nAChRs (Bertrand and Changeux, 1999). A defect in the usual α4/β2 mediated facilitation of GABA release would provide a plausible basis for epilepsy. This hypothesis would be strengthened if the described ADNFLE mutations were associated with hypofunction of α4/β2 nAChRs and if hypofunction of these receptors created a predisposition to seizures.

Functional characterisation of the ADNFLE mutations in vitro has produced an inconsistent picture. The described α4 mutations affect the second transmembrane domain of the α4 subunit (Steinlein et al., 1995, Steinlein et al., 1997, Hirose et al., 1999), while the described β2 mutations affect the corresponding domain of the β2 subunit (De Fusco et al., 2000, Phillips et al., 2001). Characterisation of the α4 S248F mutation in oocytes showed a significant decrease in ACh-induced currents and a shift in the dose–response curve indicating lower sensitivity (Steinlein et al., 1995, Kuryatov et al., 1997), whereas oocyte studies of the α4 259insL mutation revealed an increase in ACh affinity without significant changes in the total channel current (Steinlein et al., 1997, Bertrand et al., 1998). In both mutations, the ACh-evoked calcium current was reduced, which may be an important unifying observation given that calcium ions are implicated as second messengers in the neuromodulatory role of nAChRs (Lena and Changeux, 1997, Bertrand et al., 1998). However, while the above studies suggest receptor hypofunction, Figl et al. (1998) reported use-dependent potentiation of the nicotinic ACh response with these two mutations. Functional studies of the third known α4 mutation (S252L) suggest receptor hypofunction with similar affinity but faster desensitisation compared to wild type (Wt) receptors (Matsushima et al., 2002). Finally, characterisation of the described β2 mutations showed increased affinity for ACh, in the case of β2V287M, and delayed desensitisation, in the case of β2V287L, leading to increased current flow in oocyte preparations (De Fusco et al., 2000, Phillips et al., 2001).

In contrast to this divergent in vitro characterisation of the mutations, kindreds with ADNFLE have a broadly consistent phenotype (De Fusco et al., 2000, Phillips et al., 2001). This suggests that more complex models are required to determine the functional consequences of nAChR dysfunction in vivo.

To investigate the effect of extreme hypofunction of α4/β2 nAChRs on seizure threshold, mice were developed with targeted deletion of the α4 subunit (Ross et al., 2000). The behaviour of mice homozygous for this mutation was characterised in the drug-naïve state and in response to the proconvulsants pentylenetetrazole (PTZ), bicuculline, kainic acid, strychnine and 4-aminopyridine (Wong et al., 2002). Homozygous α4 Mt mice showed an enhanced seizure response with a significant excess of severe seizures and death, relative to Wt controls, in response to the GABA receptor antagonists PTZ and bicuculline. Mt mice also had an increase in kainic acid and strychnine-related minor motor seizures. Paradoxically, the seizure response to 4-aminopyridine was reduced in Mt mice. This agent causes seizures by releasing endogenous glutamate and the decreased response in Mt mice suggests that they had undergone compensatory down-regulation of glutamatergic neurotransmission.

We now report a systematic assessment of the EEG phenotype of homozygous α4 Mt mice in the drug-naïve state (during wakeful behaviour, drowsiness and sleep) and in response to the proconvulsant, PTZ.

Section snippets

Mice

Forty drug-naïve mice were studied prospectively, of which 20 were Mt and 20 were Wt. Generation of the Mt mice and their genetic background is as described previously (Ross et al., 2000). Mt mice that had been backcrossed twice to the C57BL/6 strain were used to generate 10 mating pairs of mice for interbreeding and 10 mating pairs of Wt breeders. The breeding strategy was designed to maximise heterogeneity in the genetic background of mice used in the study. Mating pairs within a genotype

Baseline EEG

Several mice (Mt mice 10, Wt mice 9) exhibited spontaneous spindles or runs of sharp waves (Fig. 1B) in the 2-h untreated, baseline period, prior to receiving any PTZ. Morphologically, these EEG features closely resembled the brief spindle episodes that were typically observed in the early phase after PTZ injection (trace 1A in Fig. 1A). The spontaneous EEG spindle events were never associated with overt behavioral arrest but frequently occurred during quiet restfulness, so a mild hypokinetic

Discussion

PTZ causes seizures by antagonising GABAA receptors (Snead et al., 1999). It binds to a site related to but distinct from the picrotoxin binding site and single channel recordings suggest that, once bound, PTZ decreases the probability of chloride channel opening without affecting open channel conduction or duration of channel opening (Huang et al., 2001).

As a proconvulsant, PTZ has been used in low-dose (10–20 mg/kg) and high-dose (50–80 mg/kg) seizure models (Maresceaux et al., 1984, Snead et

Acknowledgements

This work was supported by the National Health and Medical Research Council of Australia. J. Drago is a Monash University Logan Research Fellow. We would like to thank Jim Massalas for technical assistance.

References (39)

  • S.F. Berkovic et al.

    Autosomal dominant nocturnal frontal lobe epilepsy

  • D. Bertrand et al.

    Nicotinic receptor: a prototype of allosteric ligand-gated ion channels and its possible implications in epilepsy

  • S. Bertrand et al.

    Properties of neuronal nicotinic acetylcholine receptor mutants from humans suffering from autosomal dominant nocturnal frontal lobe epilepsy

    British Journal of Pharmacology

    (1998)
  • M. De Fusco et al.

    The nicotinic receptor β2 subunit is mutant in nocturnal frontal lobe epilepsy

    Nature Genetics

    (2000)
  • A.B. Elgoyhen et al.

    α10: A determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells

    Proceedings of the National Academy of Science USA

    (2001)
  • T.N. Ferraro et al.

    Mapping loci for pentylenetetrazol-induced seizure susceptibility in mice

    Journal of Neuroscience

    (1999)
  • A. Figl et al.

    Two mutations linked to nocturnal frontal lobe epilepsy cause use-dependent potentiation of the nicotinic ACh response

    Journal of Physiology (London)

    (1998)
  • C.J. Frazier et al.

    Synaptic potentials mediated via α-bungartotoxin-sensitive nicotinic acetylcholine receptors in rat hippocampal neurons

    Journal of Neuroscience

    (1998)
  • Y. Gioanni et al.

    Nicotinic receptors in the rat prefrontal cortex: increase in glutamate release and facilitation of mediodorsal thalamo-cortical transmission

    European Journal of Neuroscience

    (1999)
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