Alcohol and Developing Neuronal Circuits

描述（由申请人提供）：胎儿酒精暴露会导致持续的认知功能障碍，这可能是继发于海马体（涉及学习和记忆的大脑区域）损伤的继发性。在发育过程中，海马体表现出一种网络驱动的神经元活动模式，称为巨去极化电位 (GDP)。这些事件部分是由 GABAA 受体介导的，GABAA 受体在表达低水平 Cl 输出蛋白 KCC2 的未成熟神经元中去极化，因此具有较高的 [Cl]i。初步数据表明，乙醇 (EtOH) 可以有效增加 GDP 的频率，并且这种效应的潜在机制是增加中间神经元释放 GABA 和谷氨酸的可能性。我们假设 EtOH 会增加苔藓纤维中递质的释放，从而在发育中的海马体中共同释放 GABA 和谷氨酸。或者，GABA 可以从中间神经元末端释放，谷氨酸可以从锥体神经元末端释放。此外，我们假设EtOH诱导的GDP频率增加将导致[CA2]i升高，这将通过BDNF水平增加导致KCC2下调，导致GABAA受体从兴奋性到抑制性的作用延迟转换。具体目标#1 是确定乙醇诱导的 GABA 和谷氨酸增加的来源。 使用膜片钳电生理学技术和新生儿海马切片，我们将测量通过刺激苔藓纤维、中间神经元或锥体细胞在 CA3 中间神经元中诱发的 GABAA 和 AMPA 受体介导的突触后电流的配对脉冲可塑性。具体目标#2 是确定 EtOH 诱导的 GDP 频率增加是否会升高 [CA2 ]i。我们建议使用 CA2 成像和膜片钳技术来解决这种可能性，并将研究 EtOH 对发育神经元群同步程度的影响。具体目标#3 是评估 GDP 的 EtOH 调节是否会影响体外 KCC2 水平。我们还将研究 EtOH 对 BDNF 和 pCREB ​​水平的影响。具体目标#4 是 确定体内长期暴露 EtOH 对 GDP 驱动的网络活动和 KCC2 水平的影响。我们将通过吸入室将新生大鼠沿着其母鼠暴露于乙醇中，并测量 KCC2 水平的表达以及 GABAA 受体从兴奋性到抑制性的作用转变。我们还将研究体内乙醇暴露是否会引起同步神经元活动的改变。总的来说，这些研究将证实由 GABAA 兴奋作用驱动的网络神经元活动是海马发育过程中 EtOH 作用的一个新颖且重要的目标。

DESCRIPTION (provided by applicant): Fetal alcohol exposure results in persistent cognitive dysfunctions that may be secondary to damage of the hippocampus, a brain region involved in learning and memory. During development, the hippocampus displays a pattern of network-driven neuronal activity known as giant depolarizing potentials (GDPs). These events are mediated, in part, by GABAA receptors, which are depolarizing in immature neurons that express low levels of the Cl exporter, KCC2, and thus, have higher [Cl]i. Preliminary data indicates that ethanol (EtOH) potently increases the frequency of GDPs and that the underlying mechanism of this effect is an increase in the probability of GABA and glutamate release at interneurons. We hypothesize that EtOH increases transmitter release at mossy fibers, which can co-release GABA and glutamate in the developing hippocampus. Alternatively, GABA could be released from interneurons terminals and glutamate from pyramidal neuronal terminals. Moreover, we hypothesize that the EtOH-induced increase of GDP frequency will result in an elevation of [CA2+]i, which will lead to downregulation of KCC2 via an increase in BDNF levels, resulting in a delayed switch in the actions of GABAA receptors from excitatory to inhibitory. Specific Aim #1 is to determine the source of the EtOH-induced increase in GABA and glutamate. Using patch-clamp electrophyisiological techniques and neonatal hippocampal slices, we will measure paired-pulse plasticity of GABAA and AMPA receptor-mediated postsynaptic currents evoked in CA3 interneurons by stimulation of mossy fibers, internuerons, or pyramidal cells. Specific Aim #2 is to determine if the EtOH-induced increase of GDP frequency elevates [CA2+]i. We propose to use CA2+ imaging and patch-clamp techniques to address this possibilty and will also investigate the effect of EtOH on the degree of synchronization of developing neuronal ensembles. Specific Aim #3 is to assess whether EtOH modulation of GDPs affects KCC2 levels in vitro. We will also investigate the effect of EtOH on BDNF and pCREB levels. Specific Aim #4 is to determine the impact of long-term EtOH exposure in vivo on GDP-driven network activity and KCC2 levels. We will expose neonatal rats along their dams to EtOH via inhalation chambers and measure expression of KCC2 levels and the switch in the actions of GABAA receptors from excitatory to inhibitory. We will also investigate whether in vivo EtOH exposure induces alterations in synchronized neuronal activity. Collectively, these studies will establish that network neuronal activity driven by the excitatory actions of GABAA is a novel and important target of the actions of EtOH during hippocampal development.