Alcohol and Developing Neuronal Circuits

描述(申请人提供)：胎儿酒精暴露导致持续的认知功能障碍，可能继发于海马体的损害，海马体是一个涉及学习和记忆的大脑区域。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，称为巨大去极化电位(GDPs)。这些事件在一定程度上是由GABAA受体介导的，GABAA受体在未成熟神经元中去极化，表达低水平的氯输出蛋白KCC2，因此具有较高的[Cl]i。初步数据表明，乙醇(Etoh)有效地增加GDPs的频率，这种作用的潜在机制是增加中间神经元释放GABA和谷氨酸的可能性。我们假设乙醇增加苔藓纤维的递质释放，这可以在发育中的海马区共同释放GABA和谷氨酸。或者，GABA可从中间神经元终末释放，谷氨酸可从锥体神经元终末释放。此外，我们假设乙醇诱导的GDP频率增加将导致[CA2]i升高，这将通过BDNF水平的增加而导致KCC2的下调，导致GABAA受体的活动从兴奋到抑制的延迟切换。具体目标#1是确定乙醇诱导的GABA和谷氨酸增加的来源。 利用膜片钳电生理学技术和新生海马片，我们将测量GABAA和AMPA受体介导的突触后电流在刺激苔藓纤维、胞间或锥体细胞时在CA3中间神经元中的双脉冲可塑性。具体目的#2是确定乙醇诱导的GDP频率增加是否会提高[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，并将研究乙醇对发育中的神经元集合同步化程度的影响。具体目的#3是评估乙醇对GDPS的调节是否会在体外影响KCC2水平。我们还将研究乙醇对BDNF和pCREB水平的影响。具体目标#4是 确定体内长期接触乙醇对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暴露在乙醇中，并测量KCC2水平的表达和GABAA受体从兴奋到抑制的转换。我们还将研究体内乙醇暴露是否会导致同步神经元活动的改变。总之，这些研究将确定由GABAA的兴奋作用驱动的网络神经元活动是乙醇在海马区发育过程中作用的一个新的和重要的靶点。

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.