Synaptic integration and intrinsic firing properties of basal ganglia neurons

黑质多巴胺(SNC)神经元以低频率强直放电，导致动作电位反向传播。在我们实验室最近完成的一项研究中，我们发现背景紧张性放电的速度对树突定位的电导和突触驱动的放电反应产生了强大的影响。利用双光子谷氨酸切断和突触刺激，我们证明了快速紧张性放电(4-5赫兹)以NMDA受体依赖的方式增强突触驱动的猝发放电输出。此外，我们发现，将紧张率从1赫兹增加到6赫兹，显著增强了平均树突状钙信号，一些细胞显示出比线性预测的钙值高达5倍的增长。与黑质神经元相比，腹侧被盖区(VTA)多巴胺神经元表现出更为线性的钙升高，提示黑质神经元的游离钙负荷较高。这项研究已提交并已发表(Hage和Khaliq，2015)。 继续我们对研究中脑多巴胺神经元功能异质性的兴趣，我们有两个接近完成的项目，这两个项目确定了影响多巴胺神经元亚群的放电和树突兴奋性的亚阈值电导的重要差异。在第一项研究中，我们比较了投射到前额叶皮质、伏隔核和背侧纹状体的逆行标记的多巴胺神经元。通过与黑质纹状体神经元亚群的比较，我们发现VTA亚群(中皮质和中伏隔神经元)对超极化刺激后的棘波起始表现出更强的敏感性和更长的延迟。这种不同的敏感性是由于在VTA多巴胺神经元中存在一种缓慢衰减的钾电流，该电流的表达更为强烈。我们的计划是测试这一发现对多巴胺神经元亚群之间的尖峰时间和同步性的影响。在第二项研究中，我们检测了黑质多巴胺神经元的树突兴奋性。在过去的电生理学研究中，SNC神经元被认为是一个比VTA神经元更同质的群体。然而，在研究这些细胞的树突状钙信号时，我们发现黑质神经元之间阈值下反应的强度和强度存在显著差异。特别是，我们发现，很大一部分黑质多巴胺神经元对诱发和突触输入表现出非线性的去极化猝发样反应。这些非线性反应是由树突状T型钙通道驱动的，令人惊讶的是，几乎只出现在Calbindin阴性的SNC多巴胺能神经元中。这一发现具有潜在的临床意义，因为在帕金森氏症患者中，钙结合蛋白阴性的多巴胺神经元往往会选择性死亡。与我们最近发表的结果(Hage和Khaliq，2015)一起，这项研究支持了钙电导在塑造黑质多巴胺能神经元的生理过程中发挥更主导作用的观点。 在我们对多巴胺神经元的突触生理学感兴趣之后，我们一直在研究多巴胺神经元上树突棘的特性。树突存在的解剖学证据与一些观察到棘状突起的研究混杂在一起，而另一些研究则得出结论，多巴胺神经元主要是刺状的。我们发现，幼年和成年小鼠的多巴胺神经元都清楚地表达脊椎。我们已经证实，在快速灌流的组织制剂中以及高尔基体染色的组织中都存在脊柱。此外，我们使用脊椎头部的钙成像和谷氨酸去除相结合的方法测试了这一功能。这些实验表明，它们确实表达突触受体，并可能形成功能性突触。我们研究了脊柱长度对脊柱功能的影响，目前正在比较整合到棘突和整合到树突干突触上。该项目已接近完成，我们计划在不久的将来提交。最后，我们参与了一个与Ellen Sidransky博士的实验室合作的项目。最近提交了这项研究的手稿，目前正在进行审查。

Substantia nigra dopamine (SNc) neurons fire tonically at low rates resulting in action potential backpropagation. In a recently completed study from our lab, we found that the rate of background tonic firing exerts a powerful influence over dendritically-located conductances and synaptically-driven firing responses. Using two-photon glutamate-uncaging and synaptic stimulation, we show that fast tonic firing (4-5 Hz) intensifies synaptically-driven burst firing output in an NMDA-receptor dependent manner. Furthermore, we found that increasing the tonic rate from 1 to 6 Hz dramatically enhanced the average dendritic Ca signals, with some cells showing up to five-fold increase over linearly predicted Ca values. By contrast to SNc neurons, ventral tegmental area (VTA) dopamine neurons showed much more linear increases in Ca with rate suggesting a higher load of free Ca in SNc neurons. This study was submitted and has been published (Hage and Khaliq, 2015). Continuing our interest in examining functional heterogeneity among midbrain dopamine neurons, we have two projects nearing completion that identify important differences in subthreshold conductances that shape firing and dendritic excitability in subpopulations of dopamine neurons. In the first study, we compared retrogradely-labeled dopamine neurons that project to prefrontal cortex, nucleus accumbens and dorsal striatum. By comparison to SNc subpopulations (nigrostriatal neurons), we find that VTA subpopulations (mesocortical and mesoaccumbal neurons) show stronger sensitivity and longer delays to spike onset following hyperpolarizing stimuli. This differential sensitivity is due to the presence of a slowly decaying potassium current that is more strongly expressed in VTA dopamine neurons. Our plan is to test the implications of this finding on spike timing and synchrony among dopamine neuron subpopulations. In a second study, we examine dendritic excitability in SNc dopamine neurons. In past electrophysiological studies, SNc neurons were considered a much more homogeneous population than VTA neurons. Studying dendritic Ca signals of these cells, however, we have discovered dramatic differences in the strength and intensity of subthreshold responses in among SNc neurons. In particular, we find that a major fraction of SNc dopamine neurons exhibits non-linear depolarizing burst-like responses to evoked and synaptic inputs. These non-linear responses are driven by dendritic T-type Ca channels, and surprisingly, tend to be present almost exclusively in calbindin-negative SNc dopaminergic neurons. This finding has potential clinical relevance because calbindin-negative dopamine neurons tend to selectively die in Parkinsons patients. Along with our recently published results (Hage and Khaliq, 2015), this study supports the idea that Ca conductances play a much more dominant role in shaping the physiology of SNc dopaminergic neurons. Following our interest in the synaptic physiology of dopamine neurons, we have been examining the properties of dendritic spines on dopamine neurons. Anatomical evidence for the presence of dendritic spines has been mixed with some studies observing the presence of spine-like processes while others conclude that dopamine neurons are largely aspiny. We find that dopamine neurons clearly express spines in both juvenile and adult mice. We have confirmed that spines are present in fast perfused tissue preparations as well as golgi-stained tissue. Furthermore, we have tested the functionality using a combination of Ca imaging in the spine head and glutamate uncaging. These experiments show that they indeed express synaptic receptors and likely form functional synapses. We examined the effect of spine length on the function of spines and are currently comparing integration onto spines versus onto dendritic shaft synapses. This project is nearly completed and we plan to submit in the near future. Lastly, we participated in a collaborative project with the laboratory of Dr. Ellen Sidransky. A manuscript of this study was recently submitted and is under review.