Department of Psychology, SUNY Old Westbury, 223 Store Hill Road, Bldg.: NAB, Room: 2059, Old Westbury, NY, 11568-1700, USA.
SUNY Old Westbury, Neuroscience Research Institute, 223 Store Hill Road, Bldg.: NAB, Room: 2059, Old Westbury, NY, 11568-1700, USA.
J Biomed Sci. 2018 May 24;25(1):45. doi: 10.1186/s12929-018-0450-4.
Lead (Pb) is an environmental neurotoxicant that disrupts neurodevelopment, communication, and organization through competition with Ca signaling. How perinatal Pb exposure affects Ca-related gene regulation remains unclear. However, Ca 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 Pb 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- 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 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 HO. 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 Pb exposure on genes related to early neuronal development and the GABA-shift were evaluated through the expression of: Ca-β3, GABA-β3, NKCC, KCC, and GAD 80, 86, 65, and 67 isoforms.
Perinatal Pb exposure significantly altered the GABA-shift neurodevelopmental GOI expression as a function of Pb exposure and age across postnatal development. Dramatic changes were observed with Ca-β3 expression consistent with a Pb 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 Pb at PND 2 and 7, and both PFC and HP were negatively impacted by Pb at PND 22. Additionally, Pb 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 Pb at PND 22.
Perinatal Pb 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 Pb has on the GABAergic system preceding what is known regarding Pb insults unto the glutamatergic system.
铅 (Pb) 是一种环境神经毒素，通过与 Ca 信号竞争，破坏神经发育、通讯和组织。围产期 Pb 暴露如何影响与 Ca 相关的基因调控仍不清楚。然而，Ca 激活 L 型电压敏感钙通道 β-3 亚基 (Ca-β3)，它自动调节神经元兴奋性，并在 GABA 从兴奋性神经递质向抑制性神经递质的转变中发挥作用。
总共使用了 8 只雌性（n=4 只对照和 n=4 只围产期）和 4 只雄性（n=2 只对照和 n=2 只围产期）大鼠作为繁殖者，作为母鼠和父鼠。母鼠的每窝幼崽数量范围为 N=6-10 只/窝（M=8，SD=2），与 Pb 处理无关，雄性多于雌性。由于每窝雄性幼崽多于雌性，为了在研究的所有发育时间点上最好地评估和同等控制 Pb 和窝效应，由于从获得的窝中获得的雌性幼崽样本量不足，因此排除了雌性幼崽。从包括的幼崽窝中，有 24 只实验性未成熟的雄性长耳垂帽大鼠幼崽（对照 N=12；Pb N=12）用于本研究。在出生后第 2、7、14 和 22 天，从大鼠前额皮质（PFC）和海马（HP）中提取大脑，并使用玻璃-聚四氟乙烯匀浆器在 1ml TRIzol 试剂中匀浆 100mg 组织。离心后，用氯仿提取 RNA，并与异丙醇沉淀。然后将 RNA 样品重新悬浮在 100μl 的 DEPC 处理的 HO 中。接下来，用 10μg 总 RNA 用无 RNA 酶的 DNase（Qiagen）在 37°C 处理 1h，然后通过 3:1 的酚/氯仿提取，然后用乙醇沉淀进行再纯化。从纯化的 RNA 中，使用 1μg 在 SYBR GreenER 两步 qRT-PCR 试剂盒（Invitrogen）中进行第一链 cDNA 合成和定量实时 PCR（qRT-PCR）。通过评估与早期神经元发育和 GABA 转变相关的基因的表达，评估围产期 Pb 暴露对基因的影响：Ca-β3、GABA-β3、NKCC、KCC 和 GAD 80、86、65 和 67 同工型。
围产期 Pb 暴露显著改变了 GABA 转变神经发育 GOI 表达，作为 Pb 暴露和年龄在出生后发育过程中的函数。观察到 Ca-β3 表达的剧烈变化，这与 L 型钙通道与 Pb 的竞争一致。到 PND 22 时，PFC 和 HP 中的 Ca-β3 mRNA 分别减少了 1 倍和 1.5 倍，与对照组相比。所有 HP GABA-β3 mRNA 水平在 PND 2 和 7 时特别容易受到 Pb 的影响，而 PFC 和 HP 都在 PND 22 时受到 Pb 的负面影响。此外，Pb 改变了 PFC 和 HP 中的不成熟 GAD 80/86 mRNA 表达，特别是在 PND 2 时，而成熟的 GAD 65/67 在 PND 22 时受到 Pb 的影响最大。
围产期 Pb 暴露破坏了与 GABA 转变相关的 mRNAs 的表达，可能改变了神经回路在发育过程中的建立、组织和兴奋性。这些发现为 Pb 对 GABA 能系统的改变效应提供了新的见解，这些改变效应先于已知的 Pb 对谷氨酸能系统的损伤。
