Finding the projection-specific dopaminergic synaptic organizers

项目总结 从中脑到纹状体的多巴胺能投射调节各种认知和运动行为，并 与许多精神和神经退行性疾病有牵连。在解剖上和功能上有两种 不同多巴胺能投射：从黑质致密部(SNC)到尾壳核 (CPU)[中纹状体投射]和腹侧被盖区(VTA)至伏核(NAC) [中边缘投影]。中纹状体投射对运动功能至关重要，而中脑边缘投射对运动功能至关重要 投射与吸毒成瘾和情绪行为有关。然而，分子机制在- 纹状体中特定的多巴胺能突触联系的建立尚不清楚。我们 假设靶分子(CPU或NAC)在突触的建立中起关键作用 来自两个不同的多巴胺能投射。利用中脑培养，我们发现CPU提取出的 诱导SNC神经元突触前分化，而NAC提取物诱导VTA神经元突触前分化。部分提纯 结果表明，活性分子为10-30 kDa。微阵列筛选识别出不同的分子- 在多巴胺能突触形成过程中，在CPU和NAC之间有表达。其中包括BMP/ 转化生长因子β家族成员(10-30 kDa)：骨形态发生蛋白2和骨形态发生蛋白6在中央处理器中高表达，而骨形态发生蛋白3 和转化生长因子β-2。原位杂交和RT-PCR实验证实了它们的选择性表达 CPU/NAC。在培养的中脑神经元中，这些因素可以诱导多巴胺能突触前分化 以特定于投影的方式。相反，BMPR/转化生长因子β受体抑制剂可阻断CPUNAC提取物的作用 关于多巴胺能突触前分化。此外，在体内敲除这些骨形态发生蛋白/转化生长因子β导致 多巴胺能突触形成中的区域特异性(CPU或NAC)缺陷。最后，BMP2和BMP2的应用 转化生长因子β-2对中脑培养物激活不同的Smad，而不同的Smad是中央处理器和NAC所必需的 提取物依赖的多巴胺能突触前分化。我们建议靶向特异性骨形态发生蛋白/转化生长因子β 通过激活不同的Smad来调节特定多巴胺能突触连接的分化。 为了验证这一假设，我们建议：目标1：确定骨形态发生蛋白/转化生长因子β是否参与多巴胺能轴突 靶向和/或突触形成。目标2：检查BMP/的生理和行为后果 纹状体内转化生长因子β失活。目的3：探讨特异性Smad在骨形态发生蛋白/转化生长因子β介导中的作用 多巴胺能突触的体外形成。目标4：确定BMP受体和Smad在 体内投射特异性多巴胺能突触的形成。我们将使用分子细胞生物学， 生物化学、组织学、成像、电生理学和行为学方法来解决这些目标。它是 预计这项研究将揭示投射特异性多巴胺能 在哺乳动物的大脑中建立了突触连接，并将提供新的预防策略 以及治疗与多巴胺能投射有关的疾病，如物质使用和依赖。

PROJECT SUMMARY Dopaminergic projections from the midbrain to striatum regulate various cognitive and motor behaviors and are implicated in many psychiatric and neurodegenerative disorders. There are two anatomically and functionally distinct dopaminergic projections: from the substantia nigra pars compacta (SNc) to the caudate putamen (CPu) [the mesostriatal projection] and from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) [the mesolimbic projection]. The mesostriatal projection is critical for motor functions, while the mesolimbic projection is involved in drug addiction and emotional behavior. However, the molecular mechanisms under- lying the establishment of the specific dopaminergic synaptic connections in the striatum are unknown. We hypothesize that target (CPu or NAc)-specific molecules play crucial roles in the establishment of synapses from the two distinct dopaminergic projections. Using midbrain cultures, we found that CPu extract specifically induces presynaptic differentiation in SNc neurons, while NAc extract in VTA neurons. Partial purification indicated that the active molecules are 10–30 kDa. A microarray screen identified molecules that are differen- tially expressed between the CPu and NAc during dopaminergic synapse formation. Among them are BMP/ TGFβ family members (which are 10–30 kDa): BMP2 and BMP6 are highly expressed in the CPu, while BMP3 and TGFβ2 in the NAc. In situ hybridization and RT-PCR experiments confirmed their selective expressions in the CPu/NAc. In cultured midbrain neurons, these factors can induce dopaminergic presynaptic differentiation in a projection specific manner. Conversely, BMPR/TGFβR inhibitors blocked the effects of CPu/NAc extracts on dopaminergic presynaptic differentiation. Furthermore, in vivo knockdown of these BMP/TGFβ resulted in region-specific (CPu or NAc) defects in dopaminergic synapse formation. Finally, application of BMP2 and TGFβ2 to midbrain cultures activated distinct Smads, and distinct Smads were necessary for CPu and NAc extracts-dependent dopaminergic presynaptic differentiation. We propose that target-specific BMP/TGFβ regulate differentiation of specific dopaminergic synaptic connections through the activation of distinct Smads. To test this hypothesis, we propose to: Aim 1: Determine whether BMP/TGFβ contribute to dopaminergic axon targeting and/or synapse formation. Aim 2: Examine the physiological and behavioral consequences of BMP/ TGFβ inactivation in the striatum. Aim 3: Investigate the role of specific Smads in BMP/TGFβ-mediated dopaminergic synapse formation in vitro. Aim 4: Identify the BMP receptors and Smads that are critical for projection-specific dopaminergic synapse formation in vivo. We will use molecular cellular biological, biochemical, histological, imaging, electrophysiological, and behavioral approaches to address these aims. It is anticipated that this study will reveal the molecular mechanisms by which projection-specific dopaminergic synaptic connections are established in the mammalian brain, and will provide novel strategies for prevention and treatment of disorders implicated in dopaminergic projections such as substance use and dependence.