Nicotine Elicits Convulsive Seizures by Activating Amygdalar Neurons

Abstract

Nicotinic acetylcholine (nACh) receptors are implicated in the pathogenesis of epileptic disorders; however, the mechanisms of nACh receptors in seizure generation remain unknown. Here, we performed behavioral and immunohistochemical studies in mice and rats to clarify the mechanisms underlying nicotine-induced seizures. Treatment of animals with nicotine (1-4 mg/kg, i.p.) produced motor excitement in a dose-dependent manner and elicited convulsive seizures at 3 and 4 mg/kg. The nicotine-induced seizures were abolished by a subtype non-selective nACh antagonist, mecamylamine (MEC). An α7 nACh antagonist, methyllycaconitine, also significantly inhibited nicotine-induced seizures whereas an α4β2 nACh antagonist, dihydro-β-erythroidine, affected only weakly. Topographical analysis of Fos protein expression, a biological marker of neural excitation, revealed that a convulsive dose (4 mg/kg) of nicotine region-specifically activated neurons in the piriform cortex, amygdala, medial habenula, paratenial thalamus, anterior hypothalamus and solitary nucleus among 48 brain regions examined, and this was also suppressed by MEC. In addition, electric lesioning of the amygdala, but not the piriform cortex, medial habenula and thalamus, specifically inhibited nicotine-induced seizures. Furthermore, microinjection of nicotine (100 and 300 μg/side) into the amygdala elicited convulsive seizures in a dose-related manner. The present results suggest that nicotine elicits convulsive seizures by activating amygdalar neurons mainly via α7 nACh receptors.

Keywords: Fos expression; amygdala; convulsive seizures; nicotine; nicotinic acetylcholine receptors.

FIGURE 1

Effects of nicotine on convulsive seizure induction in rodents. (A,B) Nicotine-induced convulsive seizures in mice (A) and rats (B), respectively. (C) Effects of nACh antagonists, MEC (non-selective; 1 mg/kg i.p.), MLA (α7 nACh antagonist; 10 mg/kg i.p.), and DHβE (α4β2 nACh antagonist; 5 mg/kg i.p.) on nicotine (4 mg/kg. i.p.)-induced seizures in mice. Behavioral scores are expressed as the mean ± S.E.M. of 7–11 animals. Seizure incidence represents the percentage of animals, which showed convulsive seizures (score 4 or 5), against total animals examined. ^∗P < 0.05 and ^∗∗P < 0.01; Significantly different from the control animals treated with vehicle alone (Vehicle or Vehile+Vehicle). ^#P < 0.05 and ^##P < 0.01; Significantly different from the value for nicotine group (Vehicle+Nicotine).

FIGURE 2

Schematic illustrations of the brain sections selected for quantitative analysis of Fos expression. Filled squares in each section indicate the sampling areas analyzed and red squares represent the sites which showed significant increments in Fos expression by nicotine (4 mg/kg, i.p.). Anteroposterior coordinate (distance from the bregma) is shown above each section. Analysis of the MC, SC, and PirC were performed in four different levels from Bregma (Area 1 at +1.7 mm, Area 2 at +0.74 mm, Area 3 at -0.82 m, Area 4 at -2.06 mm).

FIGURE 3

Effects of nicotine (4 mg/kg, i.p.) on Fos expression in cortical regions in mice. Brains were removed 2 h after the nicotine (4 mg/kg, i.p.) administration and subjected Fos-immunochemical staining. Representative photographs illustrating the Fos-IR-positive cells in the PirC4 are shown in the left top. Each column represents the mean ± S.E.M. of 5–8 mice. ^∗P < 0.05, ^∗∗P < 0.01; Significantly different from the control animals treated with vehicle alone (Vehicle).

FIGURE 4

Effects of nicotine (4 mg/kg, i.p.) on Fos expression in subcortical regions in mice. (A) Fos expression in the limbic regions and basal ganglia. (B) Fos expression in the brain stem regions. Brains were removed 2 h after the nicotine (4 mg/kg, i.p.) administration and subjected Fos-immunochemical staining. Each column represents the mean ± S.E.M. of 5–8 mice. ^∗P < 0.05; Significantly different from the control animals treated with vehicle alone (Vehicle).

FIGURE 5

Effects of MEC on nicotine-induced Fos expression in mice. Animals were pretreated with MEC (1 mg/kg, i.p.) 15 min before the nicotine injection (4 mg/kg i.p.). Each column represents the mean ± S.E.M. of 5–8 mice. ^∗∗P < 0.01; Significantly different from the control animals treated with vehicle alone (Vehicle + Vehicle). ^#P < 0.05 and ^##P < 0.01; Significantly different from the nicotine groups (Vehicle + Nicotine).

FIGURE 6

Effects of electrical lesionings on nicotine-induced seizures in rats. Panels show the effects of nicotine (4 mg/kg, i.p.) on seizure generation in rats after electrical lesionings at the PirC (A), thalamus (Th, B), MHb (C), and amygdala (AMG, D). Right panels illustrate the electrical lesion sites in Pir, Th, MHb, and AMG. Behavioral scores (left graph) are expressed as the mean ± S.E.M. of four or five animals. Seizure incidence (right graph) represents the percentage of animals, which showed convulsive seizures (score 4 or 5), against total animals examined. ^∗P < 0.05 and ^∗∗P < 0.01; significantly different from the Sham group.

FIGURE 7

Effects of nicotine microinjected into the amygdala (AMG) on convulsive seizure induction in rats. Nicotine (100 or 300 μg/side) was locally injected into the bilateral AMG. (A) Behavioral scores (left graph) are expressed as the mean ± S.E.M. of 4–6 animals. Seizure incidence (right graph) represents the percentage of animals, which showed convulsive seizures (score 4 or 5), against total animals examined. ^∗P < 0.05 and ^∗∗P < 0.01; Significantly different from the control group treated with vehicle alone (Vehicle). (B) The injected sites of nicotine in the AMG.