MECHANISMS OF DEVELOPMENTAL LEAD NEUROTOXICITY

发育性先导神经毒性的机制

• 批准号: 2155229
• 负责人: Janusz Bogdan Suszkiw
• 金额: $ 21.72万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2155229
• 关键词: autoradiography; behavior disorders; brain septal area; choline acetyltransferase; developmental neurobiology; electrophysiology; hippocampus; immunochemistry; in situ hybridization; laboratory rat; lead poisoning; membrane channels; membrane potentials; muscarinic receptor; neurons; neurotrophic factors; newborn animals; northern blottings; nucleic acid hybridization; organ culture; receptor binding; receptor expression; scintillation spectrometry; voltage /patch clamp; voltage gated channel

The cholinergic septo-hippocampal pathway is critically involved in learning and memory functions of the hippocampus and a likely substrate for the neurotoxic effects of low-level lead (Pb) exposure in children. We observed that exposure of neonatal rats to low levels of lead causes selective reduction in ontogenic expression of cholinergic marker enzyme cholineacetyltransferase (ChAT) and muscarinic receptors in the septum without inducing similar changes in the hippocampus. This implicates the cholinergic septo-hippocampal neurons as a particularly vulnerable target for the developmental insult by inorganic lead. In this proposal we will investigate in detail, the mechanism and functional consequences of lead's effects on the septa) cholinergic cells. (1) Receptor radioligand binding to septal homogenates from control and lead-exposed neonatal rats will be used to determine the developmental time course of the Pb-induced reduction in muscarinic receptors and its persistence into adulthood; (2) Receptor subtype-selective radioligand binding and a combination of receptor autoradiography and ChAT immunocytochemistry in brain sections and/or dissociated septal cultures will be used to determine which muscarinic receptor subtype(s) are reduced and establish their localization to the cholinergic neurons; (3) a hypothesis that Pb interferes with the regulation of receptor expression by nerve growth factor (NGF) will be tested by examining the interaction between NGF and Pb on the expression of muscarinic receptors in dissociated septal cultures: (a) 125I-NGF binding to dissociated cells will be used to determine if lead interferes with binding and uptake of NGF by the target neurons and (b) receptor hybridization will be used to determine if NGF induces and Pb inhibits the induction of receptor subtypes-specific mRNA; (4) Whole cell patch-clamp recording from dissociated septal neurons in culture will be used to determine the effect of Pb exposure on the development of chemosensitivity to ACh in the septal cholinergic neurons and test Pb's effect on the electrophysiological characteristics of these neurons; specifically, we will test a hypothesis that Pb modifies the characteristic rhythmic, pacer-like firing properties of cholinergic cells by (a) attenuating muscarinic abbreviation of postspike afterhyperpolarization (AHP) , (b) altering Ca conductances, and (c) modifying the outward Ca-dependent potassium currents in the Pb-exposed cells. The phenotype of the cells will be identified after recordings by immunocytochemical staining with anti-ChAT antibodies. These experiments should provide new insights into the neurobiology of the cholinergic septo-hippocampal neurons and the extent to which alterations in their chemosensitive and electrical membrane properties might be involved in Pb-induced behavioral toxicity.

胆碱能隔s的 - 海马途径至关重要 海马和可能底物的学习和记忆功能 对于儿童低水平铅（PB）暴露的神经毒性作用。 我们观察到新生大鼠暴露于低水平的铅原因 选择性降低胆碱能标记酶的个体表达 隔膜中的cholineacetyltransferase（CHAT）和毒蕈碱受体 没有引起海马的类似变化。 这意味着 胆碱能隔膜 - 海马神经元作为特别脆弱的靶标 对于无机铅的发展侮辱。 在这个建议中，我们将 详细调查，机制和功能后果 铅对SEPTA）胆碱能细胞的影响。 （1）受体放射性物体 与对照和铅暴露的新生大鼠结合与间隔匀浆的结合 将用于确定PB诱导的发育时间课程 毒蕈碱受体的降低及其持续到成年期； （2） 受体亚型选择性放射素结合和组合 脑部的受体放射自显影和聊天免疫细胞化学 和/或解离的隔膜培养物将用于确定哪个 毒蕈碱受体亚型减少并确定其 定位于胆碱能神经元； （3）PB的假设 干扰神经生长调节受体表达 因子（NGF）将通过检查NGF和 PB关于毒蕈碱受体在解离的隔膜中的表达 培养：（a）125i-ngf与解离细胞结合将用于 确定铅是否会干扰目标的结合和吸收NGF 神经元和（b）受体杂交将用于确定NGF是否 诱导和Pb抑制受体亚型特异性mRNA的诱导； （4）从解离的间隔神经元记录的全细胞斑块钳记录 培养将用于确定PB暴露对 开发胆碱能神经元中ACH的化学敏感性 并测试PB对这些的​​电生理特征的影响 神经元；具体来说，我们将检验一个假设，PB修改了 胆碱能的特征节奏，类似起搏器的射击特性 细胞通过（a）衰减后尖头的毒蕈碱缩写 屈从于极光（AHP），（b）改变Ca电导，（C） 修改PB暴露的外向CA依赖性钾电流 细胞。 记录后，将确定细胞的表型 抗肉毒抗体的免疫细胞化学染色。 这些实验 应该提供有关胆碱能神经生物学的新见解 隔so-Hampocampal神经元及其改变的程度 化学敏感和电膜特性可能参与 PB诱导的行为毒性。