IV Prenatal Nicotine Exposure Modulates α6, α7, and α4β2 Nicotinic Acetylcholinergic Receptor Subunit Expression In Male And Female Neonatal And Adolescent Rat Offspring: An Autoradiographical Analysis
Date of Award
Open Access Dissertation
College of Arts and Sciences
Maternal smoke exposure produces long-term adverse cognitive and behavioral outcomes in offspring, including an increased likelihood of attention problems (e.g., attention deficit hyperactivity disorder; ADHD) and drug abuse. Preclinical research shows that gestational exposure to nicotine, the primary psychoactive compound in tobacco smoke, influences the neurodevelopment of attention and reward neuronal circuits. This study investigated hypotheses about five brain regions, to determine if prenatal nicotine (PN) exposure altered expression of nicotinic acetylcholine receptors (nAChRs). A low dose, intravenous nicotine (IV) exposure method was used to administer nicotine (0.05 mg/kg/injection) or saline, 3x/day on gestational days 8–21 (Treatment). Brain tissue was collected from both male and female offspring (Sex), on postnatal days (PND) 10 and 35 (Age). Autoradiography quantified [125I]-Epibatidine, [125I]α-Bungarotoxin, [125I]α-Conotoxin MII binding, to measure putative α4β2, α7, and α6-containg nAChR subunit expression levels. It was hypothesized that α4β2, α7, and α6- subunit-containing nAChRs in the Ventral Tegmental Area (VTA), Nucleus Accumbens Core (NAc), prefrontal cortex (PFC), Pendunculopontine Tegmental Nucleus (PPTg), and Hippocampus, would have significantly different expression levels between PN and prenatal saline (PS) groups (Treatment). Males were expected to have higher levels of nAChR expression across regions, and tissue from older (PND35; adolescent) subjects was hypothesized to also have higher levels of nAChR expression, compared to neonates (PND10). Following autoradiography, receptor expression levels were analyzed with the MicroBrightField software Densita, measuring receptor density as Luminance value, (a lower Luminance value indicates greater density of receptors). A total of 10 rodent dams contributed four offspring each (n=40), and the obtained Luminance values underwent preliminary statistical screening, to determine if variance analyses should proceed with Luminance as a Random Effect variable. If Litter did not have a significant effect (conservative p>0.250), a between-subjects Analysis of Variance (ANOVA) was used; if Litter had a significant effect on the shape of Luminance distribution (conservative p<0.250), statistical analyses continued with Litter as a Random Effect in a Mixed Models Linear (MML) analysis. Post-hoc analyses determined the retrospective power of each observed significant effect, based on sample size and parameter estimates derived from either ANOVA or MML. Statistically significant, sufficiently powered, main effects of Treatment were found for α4β2 nAChR expression levels in the VTA and NAcc, but no Treatment effect was found in the PPTg. As hypothesized, α7 nAChRs expression levels were lower for PN rats; the effect was only seen in the NAcc, with no significant Treatment effect found in the VTA or PFC. In the Hippocampus, Treatment was a significant main effect, but the PN group expressed more α7 nAChRs than the PS group. A statistically significant, sufficiently powered, main effect of Sex was found only in the PFC; as hypothesized, Males expressed greater density of α4β2 nAChRs. Statistically significant, sufficiently powered, main effects of Age were found for α4β2 nAChR expression in the PFC and PPTg; in the PFC, the effect was opposite: older rats expressed fewer α4β2 nAChRs. No age differences were found in the VTA or NAcc. For n α7 nAChRs, Age had a significant main effect in the Hippocampus, with adolescents expressing lower density of receptors, compared to neonates. No statistically significant, sufficiently powered effects for α6-containing nAChRs for Treatment, Sex, or Age were found. In addition to the five hypothesized regions, 12 more brain areas were analyzed for α4β2, α7, and α6-subunit containing nAChR expression levels. Those results are presented in tandem with the hypothesized findings in the Results section of this report.
Morgan, A.(2016). IV Prenatal Nicotine Exposure Modulates α6, α7, and α4β2 Nicotinic Acetylcholinergic Receptor Subunit Expression In Male And Female Neonatal And Adolescent Rat Offspring: An Autoradiographical Analysis. (Doctoral dissertation). Retrieved from http://scholarcommons.sc.edu/etd/3864