Alcohol and Developing Neuronal Circuits

描述（由申请人提供）：胎儿酒精谱系障碍（FASD）是一种以学习和记忆缺陷为特征的普遍疾病，可能是突触形成、完善和/或维持改变的结果。在之前的研究中，我们发现妊娠晚期乙醇（EtOH）暴露会改变突触成熟所必需的活性依赖的可塑性机制。在最近的这些研究中，我们证明了EtOH有效地抑制突触可塑性的一种形式，这种可塑性依赖于局部的，由l型电压门控Ca2+通道（L-VGCCs）激活的CA3锥体神经元树突的BDNF逆行释放。我们的假设是，妊娠晚期长期暴露于EtOH会持续抑制l - vgc，导致依赖于局部逆行BDNF释放的突触可塑性下降，最终导致CA3锥体神经元突触成熟延迟。目的1是验证慢性EtOH通过消耗内部Ca2+储存和STIM1结合11个亚基诱导通道降解导致持续L-VGCC抑制的假设。使用切片电生理和Ca2+成像技术，我们将评估EtOH暴露是否会导致l - vgc的持续功能抑制。我们还将使用表面生物素化试验确定EtOH暴露是否会降低L-VGCC亚基的质膜表达。使用Ca2+成像、共免疫沉淀和免疫组织化学技术，我们将研究这些影响是否是内部Ca2+储存消耗和STIM1与CaV11.2/1.3结合的结果。目的2是验证慢性EtOH抑制苔藓纤维- ca3锥体神经元突触中L-VGCC/ bdnf依赖性可塑性的假设。在这些突触中，依赖于l - vgc的BDNF逆行释放诱导了依赖于峰值时间的长时程增强，我们将使用分层电生理技术来研究这种形式的突触可塑性是否受到EtOH暴露的抑制。目的#3是通过切片电生理和免疫组织化学技术验证etoh诱导的l - vgc依赖性BDNF逆行释放抑制神经元间和MF- CA3锥体神经元突触成熟的假设。我们还将使用一种新的新生儿体内电穿孔/shRNA实验模式来确定CA3锥体神经元中L-VGCC或BDNF表达的选择性下调是否模仿慢性EtOH对这些突触发育的影响。总的来说，拟议的研究将把L-VGCC/BDNF功能障碍定义为FASD病理生理学的关键因素，为合理开发针对这种普遍疾病的治疗干预奠定基础。研究结果还将提供强有力的证据，支持即使在妊娠晚期少量饮酒也会对胎儿大脑发育产生不利影响的建议。

DESCRIPTION (provided by applicant): Fetal alcohol spectrum disorder (FASD) is a prevalent disorder characterized by learning and memory deficits that are likely a consequence of alterations in synapse formation, refinement and/or maintenance. During the previous funding period, we showed that 3rd trimester-equivalent ethanol (EtOH) exposure alters activity- dependent plasticity mechanisms that are essential for synapse maturation. In the most recent of these studies, we demonstrated that EtOH potently inhibits a form of synaptic plasticity that depends on local, retrograde release of BDNF from CA3 pyramidal neuron dendrites that is triggered by activation of L-type voltage-gated Ca2+ channels (L-VGCCs). Our hypothesis is that chronic EtOH exposure during the 3rd trimester equivalent persistently inhibits L-VGCCs, leading to a decrease in synaptic plasticity dependent on local retrograde BDNF release and ultimately causing delayed maturation of CA3 pyramidal neuron synapses. Aim #1 is to test the hypothesis that chronic EtOH causes persistent L-VGCC inhibition by inducing channel degradation via depletion of internal Ca2+ stores and STIM1 binding to 11 subunits. Using slice electrophysiological and Ca2+ imaging techniques, we will assess whether EtOH exposure causes persistent functional inhibition of L-VGCCs. We will also determine whether EtOH exposure decreases plasma membrane expression of L-VGCC subunits using a surface biotinylation assay. Using Ca2+ imaging, co- immunoprecipitation and immunhistochemical techniques, we will investigate if these effects are a consequence of depletion of internal Ca2+ stores and STIM1 binding to CaV11.2/1.3. Aim #2 is to test the hypothesis that chronic EtOH inhibits L-VGCC/BDNF-dependent plasticity at mossy fiber-CA3 pyramidal neuron synapses. At these synapses, L-VGCC-dependent retrograde release of BDNF induces spike timing- dependent long-term potentiation and we will investigate if this form of synaptic plasticity is inhibited by EtOH exposure using slice electrophysiological techniques. Aim #3 is to test the hypothesis that EtOH-induced inhibition of L-VGCC-dependent retrograde release of BDNF impairs the maturation of interneuron- and MF- CA3 pyramidal neuron synapses using slice electrophysiological and immunohistochemical techniques. We will also use a novel in vivo neonatal electroporation/shRNA experimental paradigm to determine if selective downregulation of L-VGCC or BDNF expression in CA3 pyramidal neurons mimics the effect of chronic EtOH on development of these synapses. Collectively, the proposed studies will define L-VGCC/BDNF dysfunction as a key element in the pathophysiology of FASD, forming the basis for the rational development of therapeutic interventions against this prevalent disorder. Results will also provide strong evidence supporting the recommendation that even light drinking during the 3rd trimester could adversely affect fetal brain development.