BRAIN Initiative K99 Project

BRAIN Initiative K99 项目

• 批准号: 10493925
• 负责人: Veronica A Alvarez
• 金额: $ 5.32万
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 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/10493925
• 关键词: Agonist; Attention; Automobile Driving; BRAIN initiative; Behavior; Blood - brain barrier anatomy; Blood Glucose; Brain; Brain region; Cell Nucleus; Cells; Clozapine; Complex; Consumption; Corpus striatum structure; Coupled; Dopamine; Dopamine D2 Receptor; Eating; Energy Intake; Fasting; Fire - disasters; Food; Food Intake Regulation; Globus Pallidus; Home; Hunger; Hypothalamic structure; Injections; Insulin; Insulin Receptor; Intake; Lateral; Link; Locomotion; Metabolism; Monitor; Motivation; Mus; Neurons; Nucleus Accumbens; Oxides; Pathway interactions; Periodicity; Peripheral; Population; Rewards; Rodent; Role; Saline; Satiation; Scanning; Signal Transduction; Slice; Structure of beta Cell of islet; Techniques; Training; Transfection; Viral; Water; Water consumption; Weight Gain; absorption; cell type; designer receptors exclusively activated by designer drugs; energy balance; experimental study; feeding; food consumption; hedonic; knock-down; neurochemistry; neuronal circuitry; peptide hormone; receptor

In accordance of aims of the proposal, the following experiments were done to determine the role of dopamine D2 receptor (D2R) containing neurons in the nucleus accumbens (NAc) on food intake: 1- The NAc is the ventral region of the striatum, which is a brain region implicated in reward-related behavior. In a preliminary study, Adora2a-Cre mice were injected with the Cre-dependent excitatory DREADD, hM3Dq, targeted at the nucleus NAc. This was done to specifically deliver the excitatory DREADD to the D2R-containing projection neurons in this brain region, which we could then activate using the synthetic agonist clozapine-N-oxide (CNO). Following viral introduction, mice were singly housed in specialized home cages, that continuously monitor intake of standard rodent chow and water, as well as locomotion. After four weeks of viral transfection, mice were injected with saline (5 mL/kg) for three consecutive days to habituate them to handling and injections. The final day of saline injection was used to determine baseline intake and activity, to allow for within-subjects analysis. On the fourth day mice received CNO (3 mg/kg, i.p.) and food and water intake, as well as locomotion, were recorded continuously for 24 h. Consistent with the hypothesis, activation of the NAc D2R containing neurons with peripheral administration of CNO seems to drive intake of a standard rodent chow. 2- This preliminary study relied on the activation of all D2R-containing projection neurons in the NAc, but is crucial in that it provides motivation to further explore specific subpopulations of these neurons that might be important in driving food intake. Specifically, we are examining the effects of the NAc D2R-containing neurons that project to the ventral pallidum. This is being done by injecting the retrograde CRe-dependent hM3Dq into the ventral pallidum of Adora2a-Cre mice. This will result in DREADD localization specifically in the D2R-containing neurons projecting from the NAc to the ventral pallidum, as described in my original proposal to selectively decrease the expression of D2Rs in the projection neurons of the striatum, including the NAc. D2 receptors are Gi/o-coupled receptors, which exert inhibitory control over a number of neurochemically distinct cell populations within the striatum. Therefore, removing D2Rs from the projection neurons eliminates the inhibitory actions of those receptors; this is akin to removing the break and allows the GABAergic projection neurons to more easily fire. When I selectively knocked down the D2 receptors from NAc projection neurons the mice consumed more standard rodent chow compared to littermate controls, and showed subsequent weight gain. 3. In order to further determine how the brain, and striatum specifically, might be interacting with the peripheral hunger and satiety signals, I turned my attention to insulin, a peptide hormone produced in the pancreatic beta cells. In the periphery, insulin is known to regulate metabolism by promoting the absorption of glucose from the blood. I noticed that my mice with low levels of D2 receptors on the striatal projection neurons had low fasting insulin levels compared to littermate controls (Figure 3A). Insulin produced in the periphery crosses the blood-brain barrier, and has known actions in several brain regions, including the striatum. Using qPCR techniques, I determined that mice with low levels of D2 receptors on the striatal projection neurons had increased insulin receptors in the same region. Further, this increase in striatal insulin receptors had profound effects on dopamine release. Using slice fast-scan cyclic voltammetry I replicated findings that insulin exposure leads to an increase in striatal dopamine release and found that this is amplified in mice lacking D2 receptors on the striatal projection neurons (Figure 3C). While there is still much to do, these findings begin to provide a mechanistic understanding of how the striatal circuitry might be integrating peripheral signals in the control of food ntake.

根据该提案的目的，进行了以下实验，以确定伏击核中含神经元（NAC）中含有神经元的多巴胺D2受体（D2R）在食物摄入中的作用： 1- NAC是纹状体的腹侧区域，这是与奖励相关行为涉及的大脑区域。在一项初步研究中，将adora2a-cre小鼠注入了针对Nucleus nac的Cre依赖性兴奋性兴奋性hM3DQ。这样做是为了将兴奋性恐惧剂特异性地输送到该大脑区域的含D2R投影神经元中，然后我们可以使用合成激动剂氯氮平-N-氧化物（CNO）激活该兴奋剂。病毒引入后，将小鼠单独存放在专门的家用笼子中，该笼子不断监测标准啮齿动物盘和水的摄入量以及运动。经过四个星期的病毒转染后，将小鼠连续三天注射盐水（5 mL/kg），以习惯它们进行处理和注射。盐水注射的最后一天用于确定基线摄入量和活性，以进行受试者内部分析。在第四天，将CNO（3 mg/kg，i.p.）和食物和水的摄入量以及运动记录24小时。与假设一致的是，含有CNO外周施用的NAC D2R的激活似乎驱动了标准啮齿动物盘的摄入量。 2-这项初步研究依赖于NAC中所有含D2R投影神经元的激活，但至关重要的是，它提供了进一步探索这些神经元特定亚群的动机，这些神经元可能对驱动食物摄入很重要。具体而言，我们正在研究将NAC D2R神经元投射到腹侧pallidum的影响。这是通过将逆行Cre依赖性HM3DQ注入Adora2a-Cre小鼠的腹侧粒子来完成的。如我最初的建议中所述，这将导致D2R的含D2R神经元在从NAC到腹侧颗粒的含D2R神经元中的特定定位，以选择性地降低纹状体的投影神经元中D2RS的表达，包括NAC。 D2受体是GI/O偶联受体，对纹状体内许多神经化学上不同细胞群体作用抑制性控制。因此，从投射神经元中去除D2R会消除这些受体的抑制作用。这类似于消除断裂，并使GABA能投射神经元更容易发射。当我有选择地击倒NAC投影神经元的D2受体时，与同窝对照相比，小鼠消耗了更多标准的啮齿动物盘，并显示了随后的体重增加。 3。为了进一步确定大脑和纹状体如何与外围饥饿和饱腹感信号相互作用，我将注意力转向胰岛素，这是胰岛细胞中产生的肽激素。在外围，已知胰岛素通过促进血液中的葡萄糖吸收来调节代谢。我注意到，与同窝材料对照相比，纹状体投影神经元上D2受体较低的小鼠的禁食胰岛素水平较低（图3A）。周围产生的胰岛素横穿血脑屏障，并在包括纹状体在内的几个大脑区域都有已知的作用。使用QPCR技术，我确定纹状体投影神经元上具有低水平D2受体的小鼠在同一区域的胰岛素受体增加。此外，纹状体胰岛素受体的这种增加对多巴胺释放具有深远的影响。使用切片快速扫描的循环伏安法I复制发现，胰岛素暴露会导致纹状体多巴胺释放的增加，并发现在纹状体投影神经元上缺少D2受体的小鼠中，这会得到扩增（图3C）。尽管还有很多事情要做，但这些发现开始对纹状体电路如何在控制食物的控制中整合外围信号的方式提供机械理解。