BRAIN Initiative K99 Project

根据该提案的目的，进行了以下实验以确定延髓核（NAc）中含有多巴胺D2受体（D2R）的神经元对食物摄入的作用： 1-NAc是纹状体的腹侧区域，这是一个涉及奖励相关行为的大脑区域。在一项初步研究中，Adora2a-Cre小鼠注射了靶向NAc核的Cre依赖性兴奋性DREADD hM3Dq。这样做是为了特异性地将兴奋性DREADD传递到这个大脑区域中含有D2R的投射神经元，然后我们可以使用合成激动剂氯氮平-N-氧化物（CNO）激活它。病毒引入后，将小鼠单独圈养在专门的饲养笼中，连续监测标准啮齿动物食物和水的摄入以及运动。病毒转染四周后，连续三天向小鼠注射盐水（5mL/kg）以使其习惯于处理和注射。盐水注射的最后一天用于确定基线摄入量和活动量，以便进行受试者内分析。在第四天，小鼠接受CNO（3mg/kg，i.p.）连续记录24 h的食物和水摄入量以及运动。与该假设一致，用外周施用CNO激活含有NAc D2R的神经元似乎驱动标准啮齿动物食物的摄入。 2-这项初步研究依赖于NAc中所有含D2R投射神经元的激活，但至关重要的是，它为进一步探索这些神经元的特定亚群提供了动力，这些亚群可能在驱动食物摄入方面很重要。具体来说，我们正在研究的影响NAc D2R-含有神经元的项目腹侧苍白球。这是通过将逆行性Cre依赖性hM3Dq注射到Adora2a-Cre小鼠的腹侧苍白球中来完成的。这将导致DREADD定位，特别是在含有D2R的神经元从NAc投射到腹侧苍白球，正如我最初的建议，选择性地减少D2R的表达在纹状体的投射神经元，包括NAc。D2受体是Gi/o偶联受体，其对纹状体内的许多神经化学上不同的细胞群施加抑制性控制。因此，从投射神经元中去除D2R消除了这些受体的抑制作用;这类似于去除断裂，并允许GABA能投射神经元更容易放电。当我选择性地敲低来自NAc投射神经元的D2受体时，与同窝对照组相比，小鼠消耗了更多的标准啮齿动物食物，并显示随后的体重增加。 3.为了进一步确定大脑，特别是纹状体，如何与外周饥饿和饱足信号相互作用，我将注意力转向胰岛素，一种在胰腺β细胞中产生的肽激素。在外周，已知胰岛素通过促进从血液中吸收葡萄糖来调节代谢。我注意到，与同窝对照组相比，纹状体投射神经元上D2受体水平较低的小鼠空腹胰岛素水平较低（图3A）。在外周产生的胰岛素穿过血脑屏障，并且已知在包括纹状体在内的几个脑区域中起作用。使用qPCR技术，我确定了纹状体投射神经元上D2受体水平低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增加对多巴胺的释放有深远的影响。使用切片快速扫描循环伏安法，我复制了胰岛素暴露导致纹状体多巴胺释放增加的发现，并发现这在纹状体投射神经元上缺乏D2受体的小鼠中被放大（图3C）。虽然还有很多工作要做，但这些发现开始提供了一种机制，即纹状体回路如何整合外周信号控制食物摄入。

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.