Alcohol and Developing Neuronal Circuits

描述（由申请人提供）：胎儿酒精暴露导致持续的认知功能障碍，可能继发于海马体（参与学习和记忆的大脑区域）的损伤。在发育过程中，海马体显示出一种网络驱动的神经元活动模式，称为巨大去极化电位（GDPs）。这些事件部分由GABAA受体介导，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）有效地增加了GDP的频率，这种效应的潜在机制是增加了GABA和谷氨酸在中间神经元释放的可能性。我们假设乙醇增加苔藓纤维的递质释放，苔藓纤维可以在发育中的海马中共同释放GABA和谷氨酸。另外，GABA可从中间神经元末梢释放，谷氨酸可从锥体神经元末梢释放。此外，我们假设EtOH诱导的GDP频率增加将导致[CA 2 +]i升高，这将通过BDNF水平增加导致KCC 2下调，导致GABAA受体从兴奋性到抑制性的延迟转换。具体目标#1是确定EtOH诱导的GABA和谷氨酸增加的来源。 利用膜片钳电生理技术和新生儿海马脑片，我们将测量成对脉冲可塑性的GABAA和AMPA受体介导的突触后电流在CA3中间神经元引起的苔藓纤维，internuerons，或锥体细胞的刺激。具体目标#2是确定EtOH诱导的GDP频率增加是否会升高[CA 2 +]i。我们建议使用CA 2+成像和膜片钳技术来解决这种可能性，也将研究乙醇对神经元发育的同步程度的影响。具体目标#3是评估GDPs的EtOH调节是否影响体外KCC2水平。我们还将研究EtOH对BDNF和pCREB水平的影响。具体目标#4 确定体内长期EtOH暴露对GDP驱动的网络活性和KCC2水平的影响。我们将通过吸入室将新生大鼠沿着其母体暴露于EtOH，并测量KCC 2水平的表达和GABAA受体从兴奋性到抑制性的作用转换。我们还将研究是否在体内乙醇暴露诱导同步神经元活动的改变。总的来说，这些研究将确定GABAA兴奋作用驱动的网络神经元活动是海马发育过程中EtOH作用的一个新的重要靶点。

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.