Nicotine in e-cigarette aerosol reduces GABA neuron migration via the α7 nicotinic acetylcholine receptor

Abstract

Prenatal nicotine exposure is linked to adverse neurodevelopmental outcomes, yet e-cigarette use during pregnancy continues to rise due to aggressive marketing efforts and misconceptions of safety. We investigated the effect of prenatal e-cigarette aerosol exposure on the migration of GABA neurons, a developmental process critical for the establishment of cerebral cortical circuitry. Pregnant mice were exposed to nicotine-containing aerosol (e-cigarette), nicotine-free aerosol (e-liquid) or room air (control) daily beginning 2 weeks before conception and continuing until gestational day 14. E-cigarette, but not e-liquid, aerosol significantly reduced GABA neuron density in the dorsal cerebral wall at rostral forebrain level and within the marginal zone, reflecting region-specific vulnerabilities. In vitro explant cultures revealed that nicotine dose-dependently reduced neuronal migration, and this effect was mimicked by a selective α7 nicotinic acetylcholine receptor (nAChR) agonist. Blocking the α7 nAChR using a selective antagonist attenuated the effects of nicotine on neuronal migration. These findings reveal a previously unrecognized vulnerability of GABA neuron migration to e-cigarette aerosol and identify α7 nAChR activation as a mechanism for nicotine-induced impairment of GABA neuron migration. Moreover, the findings highlight the need for translational efforts to update clinical guidance and public policy regarding e-cigarette use during pregnancy.

Keywords: Brain development; E-liquid; GAD67-GFP; Ganglionic eminence; Interneuron; Prenatal nicotine.

Fig. 1

Experimental Design. Female Swiss Webster mice were habituated to the equipment by exposure to room air for 48 or 96 min/day for 5 days. Next, they were acclimated to their exposure conditions (room air, e-liquid or e-cigarette; n = 8 per group) daily for 2 weeks. Then, the female mice were bred with unexposed GAD67-GFP^+/− heterozygous male mice. Daily exposure of the mice continued through pregnancy until gestational day 14 (GD14; day of conception was designated as GD0). Trunk blood from e-cigarette exposed dams and brains from their embryos were collected on GD14 for cotinine analysis. Brains from GAD67-GFP^+/− embryos from each of the three exposure groups were collected on GD15 (24 h. after the final room air or aerosol exposure), for analysis of GABA neuron migration in histological sections of the forebrain.

Fig. 2

GABA Cell Density in the E15 Dorsal Forebrain. Representative coronal sections at the rostral (A) and caudal (E) levels of the forebrain from a GAD67-GFP^+/− embryo showing GFP + GABA neurons (green). DAPI (blue) was used to label cell nuclei. GABA neurons were counted within a region of interest (white dashed rectangles) at each of three anatomical locations along the lateral to medial extent of the dorsal cerebral wall: lateral (a), middle (b), and medial (c). A higher magnification view of the “middle” anatomical location (b) of the dorsal cerebral wall from the rostral (B) and caudal (F) levels showing subdivision into 10 equal-sized grids for the purpose of counting GFP + GABA neurons, and stratification into marginal zone (MZ), cortical plate (CP), intermediate zone (IZ), and subventricular/ventricular zones (SVZ/VZ) (B, F). GABA neuron density is reduced in the e-cigarette group compared to the room air group at the rostral level (C) and there is no significant difference among the 3 groups at the caudal level (G). GABA neuron density was reduced in the e-cigarette group compared to the room air group in the MZ at the rostral level (D) while there were no significant differences among the groups in any lamina at the caudal level (H). Scale bars represent 300 µm (A, E) and 20 µm (B, F). * = p < 0.05 [Pair-wise comparisons with Dunnett’s (C) or Benjamini–Hochberg linear step up (D) corrections]. Data represent mean ± SEM.

Fig. 3

GABA neuron migration in explant cultures. An overview of the in vitro experimental design and outcomes (A) showing the principal steps of collection of basal forebrain eminence explants from coronal sections of E15 rostral forebrains, culturing the explants in Matrigel, and migration of GABA neurons from the explants following 48 h. in culture. In addition, a photomicrograph showing neurons migrating away from the explant edge, and the subdivision of the migration field into 80 µm-wide bins for quantitative analysis is shown. Representative micrographs showing neuronal migration under the different drug exposure conditions (B). Quantitative analyses showed that the density of GABA neurons migrating away from GE explants was reduced in the presence of 20 µM and 200 µM nicotine compared to control, whereas 2 µM nicotine did not produce significant changes (C). GABA neuron density as a percentage of the total number of migrating GABA neurons increased within the initial 80 µm of the explant edge (dashed box) following treatment with nicotine (2, 20 and 200 µM; D). The density of GABA neurons migrating from the explant was significantly reduced following treatment with the selective α7 nAChR agonist PNU282987 at 0.1 µM concentration compared to control (0.01% DMSO), whereas 1 µM or 10 µM concentrations of PNU282987 did not produce significant differences (E). None of the 3 concentrations of PNU282987 significantly altered the percentage of migrating GABA neurons located within the initial 80 µm of the explant edge compared to control (F). The density of migrating GABA neurons was significantly higher in the explants exposed to the α7 nAChR antagonist methyllycaconitine (MLA; 0.01 µM, 0.1 µM, 1 µM) 2 h. prior to nicotine exposure compared to the density in the explants exposed to nicotine (200 µM) alone, and not significantly different from the density in the control explants (G). For comparative analyses, data from explants treated with 200 µM nicotine and control explants (plain media), shown in panel C was re-incorporated. The percentage of cells within the initial 80 μm interval (dashed box) in explants treated with MLA 2 h. prior to nicotine exposure was significantly higher than the percentage in the control explants (0.01, 0.1 and 1.0 µM) and significantly lower than that in explants treated with 200 μM nicotine alone (0.1 and 1.0 µM; H). The 0.01 µM MLA treatment 2 h. prior to nicotine did not produce significant changes compared to nicotine alone (H). Pair-wise comparisons with Dunnett’s test (C, E, G) or Benjamini–Hochberg linear step-up procedure (D, H). Data represent mean ± SEM.