Neuron-glial interactions within the basal ganglia

基底神经节内神经元-胶质细胞的相互作用

• 批准号: 8736790
• 负责人: ANTONELLO BONCI
• 金额: $ 46.06万
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8736790
• 关键词: 4-Hydroxy-Tamoxifen; Ablation; Acute; Adult; Affect; Apoptosis; Basal Ganglia; Behavior; Brain; Brain region; CX3CL1 gene; Cell Density; Cell Nucleus; Cell physiology; Cells; Cellular Morphology; Childhood; Chronic; Complex; Data; Development; Diphtheria Toxin; Disease; Dose; Electric Capacitance; Exhibits; Exposure to; Future; Goals; Heterogeneity; Hippocampus (Brain); Infection; Inflammatory; Injection of therapeutic agent; Lipopolysaccharides; Medial; Membrane; Membrane Potentials; Microglia; Morphology; Mus; Neuroglia; Neurons; Nucleus Accumbens; Phagocytosis; Pharmaceutical Preparations; Play; Predisposition; Prefrontal Cortex; Property; Regimen; Regulation; Rewards; Role; Rosa; Shapes; Signal Transduction; Slice; Spinal Cord; Substantia nigra structure; Surveys; Synapses; Synaptic Transmission; Synaptic plasticity; Thick; Time; Transgenic Mice; Transgenic Organisms; Ventral Tegmental Area; Visual system structure; brain tissue; delayed rectifier potassium channel; density; diphtheria toxin receptor; drug of abuse; drug seeking behavior; information processing; pars compacta; postnatal; recombinase; release factor; research study; response; 4-Hydroxy-Tamoxifen; Ablation; Acute; Adult; Affect; Apoptosis; Basal Ganglia; Behavior; Brain; Brain region; CX3CL1 gene; Cell Density; Cell Nucleus; Cell physiology; Cells; Cellular Morphology; Childhood; Chronic; Complex; Data; Development; Diphtheria Toxin; Disease; Dose; Electric Capacitance; Exhibits; Exposure to; Future; Goals; Heterogeneity; Hippocampus (Brain); Infection; Inflammatory; Injection of therapeutic agent; Lipopolysaccharides; Medial; Membrane; Membrane Potentials; Microglia; Morphology; Mus; Neuroglia; Neurons; Nucleus Accumbens; Phagocytosis; Pharmaceutical Preparations; Play; Predisposition; Prefrontal Cortex; Property; Regimen; Regulation; Rewards; Role; Rosa; Shapes; Signal Transduction; Slice; Spinal Cord; Substantia nigra structure; Surveys; Synapses; Synaptic Transmission; Synaptic plasticity; Thick; Time; Transgenic Mice; Transgenic Organisms; Ventral Tegmental Area; Visual system structure; brain tissue; delayed rectifier potassium channel; density; diphtheria toxin receptor; drug of abuse; drug seeking behavior; information processing; pars compacta; postnatal; recombinase; release factor; research study; response

In order to define the basic properties of microglia within the basal ganglia (BG) of the adult CNS, we used CX3CR1-EGFP transgenic mice to visualize microglia within the ventral tegmental area (VTA), nucleus accumbens (NAc), substantia nigra pars compacta (SNc), and substantia nigra pars reticulata (SNr). In these transgenic mice, EGFP is expressed specifically within microglia and allows both the density and fine morphology of these cells to be quantified. Furthermore, EGFP expression allows microglia in acute brain slices to be targeted for electrophysiological recording and analysis. These analyses revealed that microglial cell properties are not uniform across distinct BG regions. Microglia within the VTA are found at significantly lower density and exhibit very sparse branching and simplified morphology compared to other BG regions. In contrast, microglia within the SNr are present at a dramatically high density and both SNr and NAc microglia display highly-ramified and complex morphology. Electrophysiological analysis revealed similar heterogeneity of microglia within these brain regions. Microglia in the VTA and the adjacent SNr differed significantly in their membrane capacitance, resting potential, and the expression of presumed delayed rectifier potassium channels. Together, these findings indicate that microglia throughout the brain cannot be considered equivalent. In addition, this heterogeneity raises important questions about whether microglia in distinct BG regions have different capacities to interact with surrounding neurons and whether their response to CNS insults will differ. Ongoing experiments include acute and chronic administration of drugs of abuse as well as lipopolysaccharide, an inflammatory agent, to probe for differential reactive responses within these cells. We have also used CX3CR1-EGFP mice to visualize microglia within the BG of the developing CNS, with a particular focus on the VTA and NAc. Quantification of microglial cell density during early postnatal periods (P6-8, P10-12, P14-16, P20-22) revealed that significant differences were present between the VTA and NAc, indicating that the regional heterogeneity observed in the adult is established shortly after microglia begin to colonize the CNS. As has been observed in other brain regions, density of microglia in the VTA and NAc peaked during the second and third postnatal weeks and then begin to taper to adult levels. However, microglial density peaked earlier in the NAc, suggesting that maturation of microglia within these two brain regions does not follow an identical time course. Dramatic changes in cell morphology across development were also evident, as microglia in both the NAc and the VTA transitioned from very simple, thick, bipolar branches to the thin, highly- ramified cell processes present in the adult. Future directions for this developmental analysis include characterization of additional brain regions, such as the medial prefrontal cortex, and experiments aimed at determining whether microglia in the early postnatal BG are engaged in phagocytosis of synapses. If microglia participate in circuit refinement within these brain regions, this raises important questions about the developmental consequences of perturbations of microglial cell function, such as childhood infections that penetrate the CNS. The data summarized above expand our understanding of microglial cells within the developing and adult BG. To begin to define whether these cells contribute to circuit refinement during development and whether they influence membrane properties and synaptic transmission of neurons within the BG of the adult, we are using transgenic strategies to specifically ablate microglia within the CNS. CX3CR1-CreER mice, which express inducible cre recombinase within microglia have been crossed to both rosa-flox-stop-diphtheria toxin (rfs-DTA) mice and rosa-flox-stop-diphtheria toxin receptor (rfs-DTR) mice. In double transgenic mice, injection of 4-hydroxytamoxifen activates Cre within microglia and those cells begin expressing DTA or DTR. In CX3CR1-CreER;rfs-DTA mice, this direct expression of diphtheria toxin within microglia causes the cells to undergo programmed cell death. In CX3CR1-CreER;rfs-DTR mice, subsequent injections with diphtheria toxin will only affect those cells expressing DTR, again pushing microglia into programmed cell death. We are currently characterizing the appropriate dose and administration regimen of 4-hydroxytamoxifen and diphtheria toxin to achieve near-complete ablation of CNS microglia and are defining the time window during which the cells remain absent from the CNS. In the future, these mice will allow us to determine whether microglia influence the basic properties of neurons in VTA and NAc and whether there are changes in the ability to induce synaptic plasticity and changes in drug-related behaviors in the absence of microglia. Furthermore, ablation of microglia during early postnatal periods will allow us to perturb microglial phagocytosis of synapses and to probe whether this results in lasting deficits in circuit function, such as a heightened susceptibility to the development of drug-seeking behaviors.

In order to define the basic properties of microglia within the basal ganglia (BG) of the adult CNS, we used CX3CR1-EGFP transgenic mice to visualize microglia within the ventral tegmental area (VTA), nucleus accumbens (NAc), substantia nigra pars compacta (SNc), and substantia nigra pars reticulata (SNr).在这些转基因小鼠中，EGFP在小胶质细胞中特别表达，并允许对这些细胞的密度和细形态进行定量。此外，EGFP表达允许将急性脑切片中的小胶质细胞用于电生理记录和分析。这些分析表明，在不同的BG区域，小胶质细胞的性质并不均匀。与其他BG区域相比，VTA内的小胶质细胞的密度明显较低，并且表现出非常稀疏的分支和简化的形态。相比之下，SNR中的小胶质细胞在很高的密度上存在，而SNR和NAC小胶质细胞都显示出高度刻板和复杂的形态。电生理分析显示，在这些大脑区域内，小胶质细胞的异质性相似。 VTA和相邻SNR中的小胶质细胞在其膜电容，静息电位以及假定延迟整流性钾通道的表达上显着差异。总之，这些发现表明，整个大脑的小胶质细胞不能被认为是等效的。此外，这种异质性提出了有关不同BG区域中的小胶质细胞是否具有不同能力与周围神经元相互作用以及它们对CNS侮辱的反应是否会有所不同。正在进行的实验包括急性和长期给药滥用药物以及炎症剂脂多糖，以探测这些细胞内反应性差异反应。 我们还使用CX3CR1-EGFP小鼠可视化发育​​中心的BG内的小胶质细胞，特别关注VTA和NAC。在产后早期（P6-8，P10-12，P14-16，P20-22）中对小胶质细胞密度的定量表明，VTA和NAC之间存在显着差异，表明在小胶质细胞开始在成年人中观察到的区域异质性在小胶质体开始在Microglia开始后不久就开始进行CNS化合物。正如在其他大脑区域观察到的那样，VTA中的小胶质细胞密度在产后和第三个几周内达到峰值，然后开始逐渐减少成人水平。然而，小胶质细胞密度在NAC早期达到峰值，表明这两个大脑区域内的小胶质细胞的成熟并不遵循相同的时间过程。 由于NAC和VTA的小胶质细胞从非常简单，厚，双极分支转变为成年人中存在的薄，高度分析的细胞过程，因此在整个发育过程中的细胞形态变化也很明显。这种发育分析的未来方向包括表征其他大脑区域，例如内侧前额叶皮层，以及旨在确定产后早期BG中的小胶质细胞是否参与突触的吞噬作用。如果小胶质细胞参与这些大脑区域内的电路细化，这将引发有关小胶质细胞功能扰动的发育后果的重要问题，例如渗透CNS的儿童感染。 上面总结的数据扩展了我们对发展中心和成人BG中小胶质细胞的理解。为了开始定义这些细胞在发育过程中是否有助于电路的细化，以及它们是否影响成年BG内神经元的膜特性和突触传播，我们正在使用转基因策略来具体灌输CNS内的小胶质细胞。在小胶质细胞中表达可诱导的CRE重组酶的CX3CR1-核小鼠已经交叉到Rosa-flox-Stop-stop-Diphtheria Toxin（RFS-DTA）小鼠和Rosa-Flox-Stop-Diphtheria毒素受体（RFS-DTR）小鼠。在双转基因小鼠中，注射4-羟基氧昔芬会激活小胶质细胞中的CRE，这些细胞开始表达DTA或DTR。在CX3CR1-CREER; RFS-DTA小鼠中，小胶质细胞中白喉毒素的这种直接表达会导致细胞发生编程的细胞死亡。在CX3CR1-CREER; RFS-DTR小鼠中，随后注射白喉毒素只会影响表达DTR的细胞，再次将小胶质细胞推入编程的细胞死亡。我们目前正在表征4-羟基莫昔芬和白喉毒素的适当剂量和给药方案，以实现CNS小胶质细胞的几乎完全消融，并定义了在CNS中不存在细胞的时间窗口。将来，这些小鼠将使我们能够确定小胶质细胞是否影响VTA和NAC中神经元的基本特性，以及在没有小胶质细胞的情况下诱导突触可塑性和药物相关行为的变化的能力是否有变化。此外，在产后早期的小胶质细胞消融将使我们能够扰动突触的小胶质细胞增多症，并探测这是否导致电路功能的持久缺陷，例如对吸毒行为发展的敏感性增强。