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: 8933870
• 负责人: ANTONELLO BONCI
• 金额: $ 40.44万
• 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/8933870
• 关键词: 4-Hydroxy-Tamoxifen; Ablation; Acute; Adult; Affect; Anxiety; Apoptosis; Astrocytes; Basal Ganglia; Behavior; Behavioral; Brain; Brain region; CSPG4 gene; CX3CL1 gene; Cell Density; Cell Nucleus; Cell Separation; Cell physiology; Cells; Cellular Morphology; Cellular biology; Childhood; Chronic; Cocaine; Corpus striatum structure; Data; Development; Diphtheria Toxin; Disease; Dorsal; Dose; Electric Capacitance; Electrons; Exhibits; Exposure to; Female; Fluorescence-Activated Cell Sorting; Future; Gene Expression; Gene Expression Profile; Genes; Goals; Heterogeneity; Hippocampus (Brain); ITGAM gene; Image; Immunohistochemistry; Infection; Inflammatory; Injection of therapeutic agent; Investigation; Macrophage Colony-Stimulating Factor Receptor; Membrane; Membrane Potentials; Messenger RNA; Methods; Microdissection; Microglia; Microscopic; Morphology; Motor Cortex; Mus; Neuroglia; Neurons; Nucleus Accumbens; Pattern; Phagocytosis; Pharmaceutical Preparations; Play; Population; Potassium; Presynaptic Terminals; Process; Property; Purinoceptor; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Rewards; Role; Rosa; Saline; Self Administration; Self-Administered; Shapes; Signal Pathway; Signal Transduction; Slice; Spinal Cord; Structure; Substantia nigra structure; Surveys; Synapses; Synaptic Transmission; Synaptic plasticity; Techniques; Tissues; Training; Transgenic Mice; Transgenic Organisms; Ventral Tegmental Area; Visual system structure; Voltage-Gated Potassium Channel; Withdrawal; brain tissue; cocaine exposure; density; diphtheria toxin receptor; dopaminergic neuron; drug of abuse; information processing; inhibitor/antagonist; interest; light microscopy; next generation sequencing; novel; oligodendrocyte precursor; pars compacta; postnatal; precursor cell; preference; recombinase; release factor; research study; response; reward circuitry; small molecule; trend; vesicular glutamate transporter 1

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). Previously we found that microglia populate the VTA at significantly lower density and exhibit sparse branching 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 morphologies. Using electrophysiological recording, we found that microglia in the VTA and the adjacent SNr differed significantly in their membrane capacitance, resting potential, and the expression of voltage-gated potassium channels. We have expanded upon these findings by examining gene expression of microglial cells isolated from distinct BG regions. Following microdissection of the VTA, SNr, NAc, dorsal striatum, and cortex from CX3CR1-EGFP mice, microglial cells were isolated from dissociated tissue using fluorescence activated cell sorting (FACS). Messenger RNA was isolated from sorted cells and RT-PCR was used to examine expression of genes of interest. The microglial-specific gene CD11b was highly expressed in FACS-isolated EGFP+ cells, while expression of the neuron-specific gene NeuN, oligodendrocyte precursor cell specific gene NG2, and astrocyte-specific gene GLT-1 was absent, indicating successful enrichment of pure populations of microglial cells. Preliminary experiments examining purinergic receptors and inflammatory signaling factors indicate that microglial expression of these genes differs across distinct BG regions. These findings suggest that there are functional differences among microglia in different regions of the BG and raise questions about whether BG microglia will exhibit variable responses to pathological insults. We have also used CX3CR1-EGFP mice to visualize microglia within the BG of mice trained to self-administer (SA) cocaine. Density, morphology, and proliferation of microglia was examined in mice that self-administered cocaine for 3 weeks as well as in mice that self-administered cocaine followed by 2 weeks of withdrawal (WD). Quantification of the % of CNS parenchyma covered by microglial processes indicated that microglial process coverage was significantly reduced in the NAc following 3 weeks SA + 2 weeks WD. Similar trends were observed for microglial cells in the VTA and SNr. These observations suggest that microglial cell morphology is altered following chronic exposure to cocaine and that these effects may be more dramatic in some BG regions than others. Changes in microglial cell morphology are correlated with alterations in the release of trophic and inflammatory signaling factors and suggest that microglial interaction with surrounding neurons may be perturbed. We are currently using more rigorous methods to quantify microglial morphology in SA and SA+WD mice. Furthermore, we are using FACS isolation of BG microglia to carry out analysis of the whole microglial transcriptome using RNAseq, a type of next generation sequencing. In future experiments, this technique will be used for unbiased examination of the microglial transcriptome in mice that have self-administered cocaine or saline. Such experiments will clarify the degree to which microglial cell biology may be altered in the absence of overt morphological reactivity and could identify novel signaling pathways that regulate interactions between microglia and neurons. Previously we used CX3CR1-EGFP mice to quantify microglial cell density and morphology during early postnatal periods and found that the regional heterogeneity observed in the adult is established early in development. To build upon this analysis, we used immunostaining for markers of presynaptic terminals and confocal imaging to determine whether microglia in the developing reward circuitry participate in synaptic pruning through phagocytosis of presynaptic terminals. Punctate structures immunolabeled for vesicular glutamate transporter 1 (VGlut1) and 2 (VGlut2) and vesicular gaba transporter (VGAT) were found colocalized with the EGFP+ processes of microglia in postnatal day 12-20 mice within the VTA and NAc. This observation suggests that microglia in the developing BG are engulfing presynaptic terminals as has been observed within the hippocampus and visual system and raises important questions about the developmental consequences of perturbations of microglial cell function, such as childhood infections that penetrate the CNS. Future experiments in this project will be aimed at determining the peak developmental period during which microglial engulfment of synapses takes place. Electron microscopic analysis will then be used to confirm observations made with immunostaining and light microscopy. The data summarized above expand our understanding of microglial cells within the developing and adult BG. To begin to define whether these cells influence the membrane properties and synaptic transmission of neurons within the BG, we are using transgenic strategies to 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. Using immunohistochemistry, we have identified a dose of 4-hydroxytamoxifen that can effect a temporary ablation of 99% of the microglia within the CNS of CX3CR1-CreER heterozygous; rfs-DTA homozygous mice. By 5 days post-ablation, microglia have repopulated 30-60% of the CNS. Preliminary electrophysiological experiments in acute brain slices from microglial-ablated mice indicate that neurons in the SNr of ablated mice have significantly reduced hyperpolarization-activated potassium current (Ih), a current which is important for regulating the firing patterns of these neurons. Ongoing experiments are aimed at determining whether similar changes in Ih occur in dopaminergic neurons of the VTA. Preliminary behavioral experiments indicate that female microglial-ablated mice have reduced anxiety-like behavior in the elevated plus maze paradigm. In addition, female microglial-ablated mice have more pronounced cocaine conditioned place preference and cocaine locomoter sensitization. Together these observations suggest that microglia interact with neurons of the basal ganglia to regulate neuronal membrane properties and BG-dependent behaviors. Future experiments will be aimed at confirming these preliminary observations and identifying possible mechanisms underlying neuron-microglial interactions in the BG. In addition, we hope to obtain a small molecule inhibitor of colony stimulating factor receptor 1 (CSFR1), manufactured by Plexikkon, that can be used to ablate microglia for long periods. Prolonged microglial ablation using this antagonist will allow investigation of mouse behavior in longer, drug self-administration paradigms.

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).以前，我们发现与其他BG区域相比，小胶质细胞以显着较低的密度填充VTA，并且表现出稀疏的分支。相比之下，SNR内的小胶质细胞存在很高的密度，而SNR和NAC小胶质细胞都显示出高度污染的形态。使用电生理记录，我们发现VTA和相邻SNR中的小胶质细胞在膜电容，静止电位以及电压门控钾通道的表达上显着差异。我们通过检查从不同BG区域分离的小胶质细胞的基因表达来扩展这些发现。在从CX3CR1-EGFP小鼠中进行了VTA，SNR，NAC，背纹状体和皮质的微分解后，使用荧光活化细胞分类（FACS）从解离组织中分离出小胶质细胞。从分类的细胞中分离出信使RNA，并使用RT-PCR检查感兴趣的基因的表达。小胶质细胞特异性基因CD11b在FACS溶解的EGFP+细胞中高度表达，而神经元特异性基因NEUN，少突胶质细胞前体细胞特异性基因NG2和星形胶质细胞特异性基因GLT-1的表达缺失，表明了纯群纯细胞的成功富集。研究嘌呤能受体和炎症信号传导因素的初步实验表明，这些基因的小胶质表达在不同的BG区域之间有所不同。这些发现表明，BG的不同区域中的小胶质细胞之间存在功能差异，并提出了有关BG小胶质细胞是否会对病理损伤表现出可变反应的问题。 我们还使用CX3CR1-EGFP小鼠来可视化受训练为自助剂（SA）可卡因的小鼠BG中的小胶质细胞。在自我管理可卡因的小鼠中检查了小胶质细胞的密度，形态和增殖，以及在自我管理的可卡因随后2周退出（WD）的小鼠中。小胶质细胞过程覆盖的CNS实质的％定量表明，在3周后NAC中，NAC的小胶质过程覆盖率显着降低。 VTA和SNR中的小胶质细胞观察到了类似的趋势。这些观察结果表明，长期暴露于可卡因后，小胶质细胞的形态发生了变化，并且某些BG区域中这些作用可能比其他效果更具戏剧性。小胶质细胞形态的变化与营养和炎症信号传导因子释放的改变相关，并表明与周围神经元的小胶质细胞相互作用可能会受到干扰。我们目前正在使用更严格的方法来量化SA+WD小鼠中的小胶质细胞形态。此外，我们使用BG小胶质细胞的FACS隔离，使用RNASEQ（一种下一代测序）对整个小胶质细胞转录组进行分析。在将来的实验中，该技术将用于对具有自我管理可卡因或盐水的小鼠中小胶质细胞转录组的无偏检查。这样的实验将阐明在没有明显的形态反应性的情况下可以改变小胶质细胞生物学的程度，并可以鉴定出调节小胶质细胞与神经元之间相互作用的新型信号通路。 以前，我们使用CX3CR1-EGFP小鼠在产后早期量化小胶质细胞的密度和形态，发现成年人在成年人中观察到的区域异质性是在发育早期建立的。为了基于此分析，我们将免疫染色用于突触前末端和共聚焦成像的标记，以确定发育中的奖励电路中的小胶质细胞是否通过突触前末端的吞噬作用参与突触修剪。对囊泡谷氨酸转运蛋白1（VGLUT1）和2（VGLUT2）和囊状GABA转运蛋白（VGAT）进行免疫标记的点状结构被与vTA和Nac内部12-20小鼠的eGFP+过程进行了共定位。该观察结果表明，正在发展中的BG中的小胶质细胞吞没了突触前末端，就像海马和视觉系统中所观察到的那样，并提出了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。该项目的未来实验将旨在确定突触的小胶质吞噬的峰值发育时期。然后，电子显微镜分析将用于确认通过免疫染色和光学显微镜做出的观察结果。 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是否影响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消化中枢神经系统内的小胶质细胞。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。使用免疫组织化学，我们已经确定了4-羟基莫昔芬的剂量，这些剂量可以影响CX3CR1-Creer杂合子CNS内99％的小胶质细胞的暂时消融； RFS-DTA纯合小鼠。到开放后5天，小胶质细胞已将30-60％的中枢神经系统重现。来自小胶质细胞驱动的小鼠的急性脑切片中的初步电生理实验表明，消融小鼠SNR中的SNR中的神经元显着降低了超极化激活的钾电流（IH），这对于调节这些神经元的发射模式很重要。正在进行的实验旨在确定VTA多巴胺能神经元中IH的类似变化。初步行为实验表明，女性小胶质细胞的小鼠在高架的迷宫范式中降低了焦虑样行为。此外，雌性小胶质细胞的小鼠具有更明显的可卡因调节位置偏好和可卡因运动敏化。总之，这些观察结果表明，小胶质细胞与基底神经节的神经元相互作用，以调节神经元膜特性和BG依赖性行为。未来的实验将旨在确认这些初步观察结果并确定BG中神经元 - 神经相互作用的可能机制。此外，我们希望获得由Plexikkon制造的菌落刺激因子受体1（CSFR1）的小分子抑制剂，可长期消化小胶质细胞。使用该拮抗剂长时间的小胶质细胞消融将允许在较长的药物自我给药范式中研究小鼠行为。