Alcohol and Developing Neuronal Circuits

描述(由申请人提供)：胎儿酒精谱系障碍(FASD)是一种普遍存在的疾病，其特征是学习和记忆障碍，这些障碍可能是突触形成、细化和/或维护改变的结果。在之前的资助期间，我们表明，第三个月相当于乙醇(Etoh)的暴露改变了突触成熟所必需的活性依赖可塑性机制。在最近的这些研究中，我们证明了乙醇有效地抑制了一种形式的突触可塑性，这种可塑性依赖于CA3区锥体神经元树突局部逆行释放脑源性神经营养因子，这种释放是由L型电压门控钙通道(L-VGCC)激活触发的。我们的假设是，在相当于妊娠晚期的慢性乙醇暴露会持续抑制L-VGCC，导致依赖于局部逆行释放脑源性神经营养因子的突触可塑性降低，最终导致CA3锥体神经元突触的延迟成熟。目的1验证慢性乙醇通过耗尽细胞内钙离子储备和STIM1与11个亚基结合而导致通道退化，从而导致持续性的L血管内皮抑制的假设。利用脑片电生理和钙离子成像技术，我们将评估乙醇暴露是否导致L-VGCCs持续的功能抑制。我们还将使用表面生物素化试验来确定乙醇暴露是否会降低L-VGCC亚单位的质膜表达。利用钙离子成像、免疫共沉淀和免疫组织化学技术，我们将研究这些效应是否是内部钙库枯竭和STIM1与CaV11.2/1.3结合的结果。目的#2验证慢性乙醇抑制苔藓纤维CA3锥体神经元突触的可塑性的假说。在这些突触上，L依赖的VGCC逆行释放脑源性神经营养因子诱导了峰时依赖性的长时程增强，我们将利用切片电生理技术来研究乙醇暴露是否抑制了这种形式的突触可塑性。目的#3用切片电生理学和免疫组织化学技术验证乙醇抑制依赖L血管内皮生长因子逆行释放的脑源性神经营养因子损害中间神经元和Mf-CA3锥体神经元突触成熟的假说。我们还将使用一种新的在体新生电穿孔/shRNA实验范式来确定选择性下调L-VGCC或脑源性神经营养因子在CA3锥体神经元中的表达是否模仿慢性乙醇对这些突触发育的影响。总之，拟议的研究将把L-血管内皮细胞癌/脑源性神经营养因子功能障碍定义为FASD病理生理学中的一个关键因素，为合理开发针对这种普遍疾病的治疗干预措施奠定基础。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.