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

Lead (Pb²⁺) is an environmental neurotoxicant that disrupts neurodevelopment, communication, and organization through competition with Ca²⁺ signaling. How perinatal Pb²⁺ exposure affects Ca²⁺-related gene regulation remains unclear. However, Ca²⁺ activates the L-Type voltage sensitive calcium channel β-3 subunit (Ca-β3), which autoregulates neuronal excitability and plays a role in the GABA-shift from excitatory-to-inhibitory neurotransmission.

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 Pb²⁺ 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 Pb²⁺- 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; Pb²⁺ 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 μL of DEPC treated H₂O. Next, 10 μg of total RNA was treated with RNase-free DNase (Qiagen) at 37 °C for 1 h and re-purified by a 3:1 phenol/chloroform extraction followed by an ethanol precipitation. From the purified RNA, 1 μg 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 Pb²⁺ exposure on genes related to early neuronal development and the GABA-shift were evaluated through the expression of: Ca-β3, GABA_AR-β3, NKCC₁, KCC₂, and GAD 80, 86, 65, and 67 isoforms.

Perinatal Pb²⁺ exposure significantly altered the GABA-shift neurodevelopmental GOI expression as a function of Pb²⁺ exposure and age across postnatal development. Dramatic changes were observed with Ca-β3 expression consistent with a Pb²⁺ competition with L-type calcium channels. By PND 22, Ca-β3 mRNA was reduced by 1-fold and 1.5-fold in PFC and HP respectively, relative to controls. All HP GABA-β3 mRNA levels were particularly vulnerable to Pb²⁺ at PND 2 and 7, and both PFC and HP were negatively impacted by Pb²⁺ at PND 22. Additionally, Pb²⁺ altered both the PFC and HP immature GAD 80/86 mRNA expression particularly at PND 2, whereas mature GAD 65/67 were most significantly affected by Pb²⁺ at PND 22.

Perinatal Pb²⁺ exposure disrupts the expression of mRNAs related to the GABA-shift, potentially altering the establishment, organization, and excitability of neural circuits across development. These findings offer new insights into the altered effects Pb²⁺ has on the GABAergic system preceding what is known regarding Pb²⁺ insults unto the glutamatergic system.