Novel Seizure Phenotype and Sleep Disruptions in Knock-In Mice with Hypersensitive α4^*Nicotinic Receptors

Abstract

A leucine to alanine substitution (L9ЈA) was introduced in the M2 region of the mouse ␣4 neuronal nicotinic acetylcholine receptor (nAChR) subunit. Expressed in Xenopus oocytes, ␣4(L9ЈA)␤2 nAChRs were Ն30-fold more sensitive than wild type (WT) to both ACh and nicotine. We generated knock-in mice with the L9ЈA mutation and studied their cellular responses, seizure phenotype, and sleepwake cycle. Seizure studies on ␣4-mutated animals are relevant to epilepsy research because all known mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) occur in the M2 region of ␣4 or ␤2 subunits. Thalamic cultures and synaptosomes from L9ЈA mice were hypersensitive to nicotine-induced ion flux. L9ЈA mice were ϳ15-fold more sensitive to seizures elicited by nicotine injection than their WT littermates. Seizures in L9ЈA mice differed qualitatively from those in WT: L9ЈA seizures started earlier, were prevented by nicotine pretreatment, lacked EEG spike-wave discharges, and consisted of fast repetitive movements. Nicotine-induced seizures in L9ЈA mice were partial, whereas WT seizures were generalized. When L9ЈA homozygous mice received a 10 mg/kg nicotine injection, there was temporal and phenomenological separation of mutant and WT-like seizures: an initial seizure ϳ20 s after injection was clonic and showed no EEG changes. A second seizure began 3-4 min after injection, was tonic-clonic, and had EEG spike-wave activity. No spontaneous seizures were detected in L9ЈA mice during chronic video/EEG recordings, but their sleep-wake cycle was altered. Our findings show that hypersensitive ␣4* nicotinic receptors in mice mediate changes in the sleep-wake cycle and nicotineinduced seizures resembling ADNFLE.

Chronic Nicotine Cell Specifically Upregulates Functional α4* Nicotinic Receptors: Basis for Both Tolerance in Midbrain and Enhanced Long-Term Potentiation in Perforant Path

“…The strategy used to generate the ␣4YFP line adapts the procedures used to generate the previous Leu9ЈSer line (Labarca et al, 2001;Tapper et al, 2004;Fonck et al, 2005) (see Fig. 1 A-D).…”

“…Changes in [Ca 2ϩ ] i were measured in ventral midbrain neuronal cultures using the ratiometric fura-2 method as described previously (Nashmi et al, 2003;Tapper et al, 2004;Fonck et al, 2005). Ventral midbrain tissue [including the substantia nigra (SN) and ventral tegmental area (VTA)] obtained from embryonic day 14 embryos was digested with 1 mg/ml papain, and cells were mechanically separated by gentle pipetting.…”

“…Cultures were prepared and maintained as described in the fura-2 experiments. Whole-cell recordings were performed with glass electrodes (2-5 M⍀) filled with an internal solution [in mM: 88 KH 2 PO 4 , 4.5 MgCl 2 , 0.9 EGTA, 9 HEPES, 0.4 CaCl 2 , 14 creatine phosphate, 4 Mg-ATP, and 0.3 GTP (Tris salt), pH 7.4 with KOH] (Nashmi et al, 2003;Fonck et al, 2005). Channel blockers to suppress nonspecific responses were included in the extracellular solution (in M: 0.5 TTX, 0.5 atropine, 10 CNQX, 20 bicuculline, 50 AP-5, and 0.01 MLA).…”

Nicotine-Induced Dystonic Arousal Complex in a Mouse Line Harboring a Human Autosomal-Dominant Nocturnal Frontal Lobe Epilepsy Mutation

“…Adult 9Ј knock-in mice do not display spontaneous seizures (Fonck et al, 2003(Fonck et al, , 2005 but are hypersensitive to many effects of nicotine. The reported L9ЈA mutant low-dose nicotine-induced "behavioral seizures" are not associated with epileptiform spike-and-wave electroencephalographic (EEG) changes (Fonck et al, 2005); in this respect, the L9ЈA nicotineinduced behavior resembles most ADNFLE attacks (Combi et al, 2004).Two ADNFLE knock-in strains have been described: the Chrna4S252F (same as our S248F M2-6Ј mutation) and the ␣4 in-frame insertion of a leucine at M2-17Ј (Klaassen et al, 2006). Both lines display spontaneous seizures of two types: brief periods of behavioral arrest and extended attacks of wild running associated with falls.…”

Genetics of nicotinic acetylcholine receptors: Relevance to nicotine addiction

“…To date, research into the role of human nAChR polymorphisms in smoking behavior has not resulted in significant associations, but this may be because functional alterations in nAChRs results in more profound dysfunction, such as nocturnal frontal lobe epilepsies [146,147] for a review see [148]. Limited power analyses, population variations and variable phenotypic definitions of nicotine dependence may also contribute to the lack of progress in the human genetics of vulnerability to smoking.…”