BRAIN Initiative K99 Project

根据该提案的目的，进行了以下实验，以确定伏核(NAC)中含有多巴胺D2受体(D2R)的神经元在摄食中的作用： 1-NAC是纹状体的腹侧区域，这是一个与奖赏相关行为有关的大脑区域。在一项初步研究中，Adora2a-CRE小鼠被注射了依赖CRE的兴奋性DREADD，hM3Dq，靶向Nac核。这是为了将兴奋性DREADD特异性地传递到该脑区含有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。在病毒感染后，小鼠被单独关在专门的家庭笼子里，这些笼子持续监测标准啮齿动物食物和水的摄入量，以及运动情况。病毒转染四周后，小鼠连续三天注射生理盐水(5mL/kg)，使其习惯于处理和注射。注射生理盐水的最后一天被用来确定基线摄入量和活动度，以便进行受试者内部分析。第4天给小鼠灌胃CNO(3 mg/kg，i.p)。连续24小时记录食物和水的摄入量以及运动。与假设一致的是，外周注射CNO激活含有NAC D2R的神经元似乎推动了标准啮齿动物食物的摄取。 2-这项初步研究依赖于NAC中所有含有D2R的投射神经元的激活，但它至关重要，因为它提供了进一步探索这些神经元的特定亚群的动力，这些亚群可能对推动食物摄入很重要。具体地说，我们正在研究投射到腹侧苍白球的含有NAC D2R的神经元的影响。这是通过将逆行依赖Cre的hM3Dq注射到Adora2a-Cre小鼠的腹侧苍白球中来完成的。这将导致DREADD定位于从NAC投射到腹侧苍白球的D2R神经元，正如我最初提出的选择性减少D2R在纹状体投射神经元(包括NAC)中的表达所描述的那样。D2受体是Gi/o偶联受体，对纹状体内许多神经化学上不同的细胞群起抑制控制作用。因此，从投射神经元中移除D2Rs就会消除这些受体的抑制作用；这类似于移除断裂，从而使GABA能投射神经元更容易放电。当我选择性地击倒NAC投射神经元中的D2受体时，与产仔鼠对照组相比，小鼠摄入了更多的标准啮齿动物食物，并显示出随后的体重增加。 3.为了进一步确定大脑，特别是纹状体，可能会如何与周围的饥饿和饱腹感信号相互作用，我将注意力转向了胰岛素，这是一种在胰腺β细胞中产生的多肽激素。在外周，胰岛素通过促进血液中葡萄糖的吸收来调节新陈代谢。我注意到，我的纹状体投射神经元上D2受体水平较低的小鼠与产仔对照组相比，其空腹胰岛素水平也较低(图3A)。外周产生的胰岛素可以穿过血脑屏障，并在包括纹状体在内的几个大脑区域发挥作用。使用qPCR技术，我确定纹状体投射神经元上D2受体水平较低的小鼠，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，并发现这种情况在纹状体投射神经元上缺乏D2受体的小鼠身上放大(图3C)。虽然仍有许多工作要做，但这些发现开始提供了对纹状体回路如何整合外周信号来控制食物摄取的机械性理解。

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.