Perinatal Pb2+ exposure alters the expression of genes related to the neurodevelopmental GABA-shift in postnatal rats

Background Lead (Pb.sup.2+) is an environmental neurotoxicant that disrupts neurodevelopment, communication, and organization through competition with Ca.sup.2+ signaling. How perinatal Pb.sup.2+ exposure affects Ca.sup.2+-related gene regulation remains unclear. However, Ca.sup.2+ activates the L-Type voltage sensitive calcium channel [beta]-3 subunit (Ca-[beta]3), which autoregulates neuronal excitability and plays a role in the GABA-shift from excitatory-to-inhibitory neurotransmission. Method A total of eight females (n = 4 Control and n = 4 Perinatal) and four males (n = 2 Control and n = 2 Perinatal) rats were used as breeders to serve as Dams and Sires. The Dam's litters each ranged from N = 6-10 pups per litter (M = 8, SD = 2), irrespective of Pb.sup.2+ treatment, with a majority of males over females. Since there were more males in each of the litters than females, to best assess and equally control for Pb.sup.2+- and litter-effects across all developmental time-points under study, female pups were excluded due to an insufficient sample size availability from the litter's obtained. From the included pup litters, 24 experimentally naïve male Long Evans hooded rat pups (Control N = 12; Pb.sup.2+N = 12) were used in the present study. Brains were extracted from rat prefrontal cortex (PFC) and hippocampus (HP) at postnatal day (PND) 2, 7, 14 and 22, were homogenized in 1 mL of TRIzol reagent per 100 mg of tissue using a glass-Teflon homogenizer. Post-centrifugation, RNA was extracted with chloroform and precipitated with isopropyl alcohol. RNA samples were then re-suspended in 100 [mu]L of DEPC treated H.sub.2O. Next, 10 [mu]g of total RNA was treated with RNase-free DNase (Qiagen) at 37 [degrees]C for 1 h and re-purified by a 3:1 phenol/chloroform extraction followed by an ethanol precipitation. From the purified RNA, 1 [mu]g was used in the SYBR GreenER Two-Step qRT-PCR kit (Invitrogen) for first strand cDNA synthesis and the quantitative real-time PCR (qRT-PCR). The effects of perinatal Pb.sup.2+ exposure on genes related to early neuronal development and the GABA-shift were evaluated through the expression of: Ca-[beta]3, GABA.sub.AR-[beta]3, NKCC.sub.1, KCC.sub.2, and GAD 80, 86, 65, and 67 isoforms. Results Perinatal Pb.sup.2+ exposure significantly altered the GABA-shift neurodevelopmental GOI expression as a function of Pb.sup.2+ exposure and age across postnatal development. Dramatic changes were observed with Ca-[beta]3 expression consistent