Prenatal Nicotine Exposure Impairs the Proliferation of Neuronal Progenitors, Leading to Fewer Glutamatergic Neurons in the Medial Prefrontal Cortex

Abstract

Cigarette smoking during pregnancy is associated with various disabilities in the offspring such as attention deficit/hyperactivity disorder, learning disabilities, and persistent anxiety. We have reported that nicotine exposure in female mice during pregnancy, in particular from embryonic day 14 (E14) to postnatal day 0 (P0), induces long-lasting behavioral deficits in offspring. However, the mechanism by which prenatal nicotine exposure (PNE) affects neurodevelopment, resulting in behavioral deficits, has remained unclear. Here, we report that PNE disrupted the proliferation of neuronal progenitors, leading to a decrease in the progenitor pool in the ventricular and subventricular zones. In addition, using a cumulative 5-bromo-2'-deoxyuridine labeling assay, we evaluated the rate of cell cycle progression causing the impairment of neuronal progenitor proliferation, and uncovered anomalous cell cycle kinetics in mice with PNE. Accordingly, the density of glutamatergic neurons in the medial prefrontal cortex (medial PFC) was reduced, implying glutamatergic dysregulation. Mice with PNE exhibited behavioral impairments in attentional function and behavioral flexibility in adulthood, and the deficits were ameliorated by microinjection of D-cycloserine into the PFC. Collectively, our findings suggest that PNE affects the proliferation and maturation of progenitor cells to glutamatergic neuron during neurodevelopment in the medial PFC, which may be associated with cognitive deficits in the offspring.

Figure 1

PNE reduced the density of glutamatergic neurons in the medial PFC. (a) Glutamate and GABA contents in the frontal cortex of control (n=14 at the age 10–12 weeks consists of seven males and seven females) and PNE prenatal nicotine exposure mice (n=12 at the age 10–12 weeks consists of five males and seven females). (b) Basal extracellular glutamate level and the high potassium (50 mM) stimulation-evoked glutamate release were measured in the PFC of control (n=8 at the age 12–16 weeks consists of four males and four females) and PNE mice (n=8 at the age 12–16 weeks consists of four males and four females). (c) NeuN- and GLS-immunostained coronal sections of the medial PFC in control and PNE mice. Quantitative analysis of the density of NeuN- and NeuN/GLS-stained cells was shown (n=11 for control mice at the age 10–12 weeks consists of four males and seven females, n=11 for PNE mice at the age 10–12 weeks consists of four males and seven females). Values are the means±SEM. *P<0.05, **P<0.01 vs corresponding control mice Scale bar: 100μm.

Figure 2

PNE impaired neurogenesis but not neuronal migration. (a, b) PNE did not affect laminar destination of cells generated on E13–E16 in the P7 or adult. Fluorescence images of BrdU⁺ cells and distribution of BrdU⁺ cells, labeled on E13–E16, in the medial PFC of control and PNE mice at each distance from the pial surface are shown (n=6–8 for control mice consists of 3–5 males and 3–4 females, n=6–8 for PNE mice consists of 3–5 males and 3–4 females). To label mitotic progenitors in the embryonic brains, the pregnant mice were given a single BrdU injection on E13, E14, E15, or E16, and then their offspring brains were collected on (a) postnatal day 7 or (b) adult at the age 8–10 weeks. (c) Quantitative analysis of the density of BrdU⁺ cells is shown (n=6–8 for control mice consists of 3–5 males and 3–4 females, n=6–8 for PNE mice consists of 3–5 males and 3–4 females). **P<0.01 vs corresponding control mice. (d) Reduction of BrdU⁺ cells in the proliferative zones that migrate to form the medial PFC of PNE mice was blocked by nAChRs α7 antagonist, but not α4β2 antagonist. Pregnant mice were given a single BrdU injection 15 min after pretreatment with MLA or DHβE on E14, and then were killed 30 min later. Fluorescence images of BrdU⁺ cells in the proliferative zones that migrate to form the medial PFC are shown. Quantitative analysis of the density of BrdU⁺ cells in control and PNE mice pretreated with MLA or DHβE are shown (n=3–6 for control mice, n=6–8 for prenatal nicotine exposure mice). Values are the means±SEM of at least three independent experiments. *P<0.05 vs SAL-treated control mice, ^##P<0.01 vs SAL-treated PNE mice. LV: lateral ventricle. Scale bar: 100 μm.

Figure 3

PNE reduced the density of neuronal progenitors in the VZ and SVZ. On E14, E16, or E18 mice were decapitated during nicotine exposure. The pups were analyzed by immunohistochemical analysis using anti-BrdU antibody as a marker of proliferarion, anti-Tbr2 antibody as a marker for intermediate progenitors in the SVZ, anti-Pax6 antibody as a marker for neuronal progenitors in the VZ, anti-PCNA as a marker of proliferation, and anti-pH3 as a marker of mitosis. Typical images of BrdU-, Tbr2-, Pax6-, PCNA-, and pH3-positive cells are shown on the left side of panels. Values are the means±SEM of at least three independent experiments (n=6–7 for control mice, n=6–7 for PNE mice). **P < 0.01 vs control mice. LV: lateral ventricle. Scale bar: 100 μm.

Figure 4

PNE-induced cognitive deficits were ameliorated by treatment with DCS in adulthood. (a, b) PNE mice exhibited cognitive deficits in the latent inhibition and fear conditioning tests in adulthood. (a) Freezing to tone under a 4CS-US protocol in the latent inhibition test was measured 24 h after the conditioning (n=10 for control mice at the age 10–12 weeks consists of four males and six females, n=11 for PNE mice at the age 10–12 weeks consists of four males and seven females). **P<0.01 vs corresponding NPE group, ^##P<0.01 vs PE-control group. (b) Freezing to tone in the conditioning (day 1), extinction (day 2), and recall (day 3) phases were measured in the fear conditioning test (n=7–16 for control mice at the age 10–12 weeks consists of 5–8 males and 2–8 females, n=9–17 for PNE mice at the age 10–12 weeks consists of 7–10 males and 2–7 females). **P<0.01 vs corresponding groups, ^##P<0.01 vs control group. (c–e) Systemic administration of DCS ameliorated behavioral deficits in PNE mice (n=18 for control mice at the age 10–14 weeks consists of 6–9 males and 9–12 females, n=16 for PNE mice at the age 10–14 weeks consists of 8–10 males and 6–8 females). DCS (30 mg/kg, s.c.) was administered to the PNE mice 30 min before these behavioral experiments. (c) Object-based attention test. Exploratory preference (left side panel) and total approach time (right side panel) were measured during a 3-min session. (d) Marble-burying test. Hidden marbles were counted during a 15-min session. (e) Elevated plus-maze test. Open arm entries (left side panel) and total arm entries (right side panel) were measured during a 5-min session. **P<0.01 vs SAL-treated control mice, ^##P<0.01 vs SAL-treated PNE mice. (f, g) Microinjections of DCS into the PFC ameliorated cognitive deficits but had little effect on the anxiety-like behavior in PNE mice (n=11–12 for control mice at the age 12–16 weeks consists of 5–6 males and six females, n=10 for PNE mice at the age 12–16 weeks consists of five males and five females). (f) In the fear conditioning test, DCS (5 nmol per 0.5 μl/bilaterally) was injected 10 min before the extinction trials. (g) In the elevated plus-maze test, DCS (5 nmol per 0.5 μl/bilaterally) was injected mice 10 min before the beginning of the test. **P<0.01 vs SAL-treated control mice, ^##P < 0.01 vs SAL-treated PNE mice. Values are the means±SEM. Abbreviations: CS, conditioned stimulus; No-Ext, No-extinction; NPE, non-pre-exposed; N.S., not significant; PE, pre-exposed; US, unconditioned stimulus.