Increased sensitivity to agonist-induced seizures, straub tail, and hippocampal theta rhythm in knock-in mice carrying hypersensitive alpha 4 nicotinic receptors

Abstract

We studied a strain of exon replacement mice ("L9'S knock-in") whose alpha4 nicotinic receptor subunits have a leucine to serine mutation in the M2 region, 9' position (Labarca et al., 2001); this mutation renders alpha4-containing receptors hypersensitive to agonists. Nicotine induced seizures at concentrations (1 mg/kg) approximately eight times lower in L9'S than in wild-type (WT) littermates. At these concentrations, L9'S but not WT showed increases in EEG amplitude and theta rhythm. L9'S mice also showed higher seizure sensitivity to the nicotinic agonist epibatidine, but not to the GABA(A) receptor blocker and proconvulsant bicuculline. Dorsiflexion of the tail (Straub tail) was the most sensitive nicotine effect found in L9'S mice (0.1 mg/kg). The L9'S mice were hypersensitive to galanthamine- and tacrine-induced seizures and Straub tails. There were no apparent neuroanatomical differences between L9'S and WT mice in several brain regions. [(125)I]Epibatidine binding to brain membranes showed that the mutant allele was expressed at approximately 25% of WT levels, presumably because of the presence of a neomycin selection cassette in a nearby intron. (86)Rb efflux experiments on brain synaptosomes showed an increased fraction of function at low agonist concentrations in L9'S mice. These data support the possible involvement of gain-of-function alpha4 receptors in autosomal dominant nocturnal frontal-lobe epilepsy.

Fig. 1.

Nicotine induction of seizures and Straub tail in L9′S and WT littermate mice. A, B, Animals received a single subcutaneous nicotine injection, and the percentage of mice that showed seizures and the time of seizure onset were recorded. C, D, Animals used in A were also scored for Straub tail. The percentage of mice responding and the time of Straub onset were recorded. Continuous video recording of the mice after nicotine injection lasted 430 sec. Animals that did not respond during video recording time were assigned a 430 sec onset time. Each data point from the onset graphs is the mean ± SE; n = 6.E, Comparison of percentage responses for Straub tail and seizure in L9′S mice, showing the higher sensitivity to Straub tail. F, L9′S animals received either a saline or a 2 mg/kg intraperitoneal mecamylamine injection, followed by a 1.5 mg/kg subcutaneous nicotine injection 5 min later. Time to seizure and Straub tail were recorded. Each bar represents mean ± SE;n = 5. The horizontal line indicates the maximum observation period (600 sec). None of the mecamylamine plus nicotine-treated mice had seizures, and 60% displayed Straub tail during this period.

Fig. 2.

Epibatidine, bicuculline, galanthamine, or tacrine induction of seizures (left panels) and Straub tail (right panels) in L9′S and WT littermate mice.A, Animals received a single subcutaneous epibatidine injection, and the times of seizure and/or Straub tail onset were recorded. B, Animals received a single bicuculline intraperitoneal injection, and the times of seizure and/or Straub tail onset were recorded. C, Animals received a single intraperitoneal galanthamine injection, and the times of seizure and/or Straub tail onset were recorded. D, Animals received a single subcutaneous tacrine injection, and the times of seizure and/or Straub tail onset were recorded. Mice with seizures lasting ≥5 min were killed. Animals injected with epibatidine or bicuculline that did not respond during video-recording time were assigned a 350 sec onset time. Animals injected with galanthamine that did not respond were assigned a 450 sec onset time, and nonresponding mice treated with tacrine were assigned a 600 sec onset time. Each data point is the mean ± SE; n = 4 or 6. There was a significant difference in seizure onset between WT and L9′S mice injected with 40 or 80 mg/kg tacrine (p < 0.05).

Fig. 3.

Morphine and nicotine induction of Straub tail in L9′S mice. Animals received a subcutaneous morphine or nicotine injection 10 min after an intraperitoneal naloxone or saline injection. Morphine and nicotine induced Straub tail in L9′S mice.A, Morphine Straub induction was blocked by the μ-opioid receptor antagonist naloxone. *p < 0.01 when comparing saline- and naloxone-treated groups (ttest; n = 4). B, Nicotine-induced Straub was unaffected by naloxone. Experiments lasted 30 min, and animals not responding within that time received a 30 min onset score. Each bar represents mean ± SE; n = 4.

Fig. 4.

Traces from hippocampal field recordings and power spectrum analysis in WT (A) and L9′S (B) mice, respectively, before and after a 2 mg/kg nicotine injection. Raw traces and power spectra analysis reveal an increase in peak-to-peak signal and theta rhythm amplitude, respectively, during seizure in the L9′S animal. C, A typical WT mouse, before and 10 min after a 10 mg/kg nicotine injection. Each power spectrum represents 1 min of continuous data.D, Box-plot graph showing a significant increase in the SD of L9′S traces (n = 6 animals) injected with 2 mg/kg nicotine or WT mice injected with 10 mg/kg nicotine (n = 5) compared with WT mice (n = 3) injected with 2 mg/kg nicotine (*p = 0.05). There was no behavioral or electroencephalographic evidence of seizure in any wild-type mouse injected with nicotine (2 mg/kg).

Fig. 5.

Nissl-stained coronal brain sections of WT (left panels) and L9′S (right panels) mice. A, Frontal cortex shown at the level of the primary motor cortex. Layers are labeled. B, Ventrolateral aspect of the thalamus. VL, Nucleus ventralis lateralis thalami; VP, nucleus ventralis posterior thalami; RT, nucleus reticularis.C, Hippocampus and adjacent secondary visual cortex.D, Dorsomedial aspect of the hindbrain, including area postrema (AP) and the hypoglossal nucleus (12).

Fig. 6.

[¹²⁵I]Epibatidine binding to mouse brain membranes. A, Epibatidine binding in membranes prepared from adult WT and heterozygous (Het) L9′S brains. Each data point is the mean ± SE;n = 4. B, Epibatidine binding in membranes obtained from WT, heterozygous, and homozygous (Hom) embryonic brains. Each data point is the mean ± SE; n = 4. C, D, Cytisine-sensitive and cytisine-resistant epibatidine binding, respectively, in embryonic membranes. Each data point is the mean ± SE; n = 4; *p < 0.01.

Fig. 7.

Rubidium efflux from synaptosomes prepared from adult WT (■) and heterozygous L9′S (●) mouse brains. In each case, the curves represent fits to two components of efflux. The EC₅₀ values (K1, K2) and maximal efflux (V1, V2) are given in theboxes. A, Acetylcholine induced rubidium efflux. Each data point is the mean ± SE; n = 5. B, Nicotine (Nic)-induced rubidium efflux. Each data point is the mean ± SE; n = 5. Het, Heterozygous.

Fig. 8.

Expected types and proportions of L9′S and WT α4β2 receptors in L9′S mice. The α4L9′S subunit is abbreviated α4*. The x-axis is the proportion of hypersensitive subunits, x = α4*/(α4* + α4), and runs from 0 to 1. We assume that the complete multimeric receptor has the stoichiometry (α4)₂(β2)₃, although the three non-α subunits may have a more complex identity. The proportion of receptors with two WT α4 subunits, 1 −x², is shown in green and decreases along the x-axis. The proportion of receptors with one mutant and one WT subunit, x(l −x), is shown in black and peaks atx = 0.5. The proportion of receptors with two mutated subunits, x², is shown inred and increases along thex-axis. The area inside the green rectangle represents the gene dosage of hypersensitive subunit that allows viable mice. This area extends from 0 to x = 0.2 = 0.25/(1 + 0.25), representing full expression of the normal allele and 25% expression of the mutant allele, as found in the present study. The value x = 0.5 would represent the lethal neo-deleted heterozygote, and x = 1 would represent the lethal neo-deleted and neo-intact homozygotes (Labarca et al., 2001).