Unraveling hippocampal networks related to food memories

第一个实验将使用免疫组织化学方法研究MC4Rs和Leprs在海马区的重叠/非重叠表达。下丘脑的这两组神经元都编码饱腹感信号，并通过不同的信号级联和电路机制起到钝化进食的作用。尽管这两种受体都没有良好的抗体，而且由于低水平的表达，在原位几乎是不可能的，但我们可以在注射瘦素后将来自Mc4r启动子驱动的Cre系的GFP信号与STAT3表达共定位(参见Myers等人2012年)。 接下来，明显的实验是用ChR2或eNpHR转导Lepr+和/或Mc4r+海马神经元，并评估光抑制的光刺激对摄食(数量和进食模式，包括进食大小、进食频率、进食长度)和体重的影响。如果我们在行为上看到一些令人信服的变化，那么我们就再次处于一个很好的位置，可以使用病毒式工具来剖析关于输入和输出的电路。 更有趣的是在光基因操作过程中执行条件性位置偏好和其他旨在获取食物的记忆/觅食任务的能力。我们可以使用简单的小室进行这些操作，甚至可以使用专门的Phenotyper笼子设计自动化方案。 与上面列出的其他项目一样，我们可以(也肯定应该)使用体外和体外切片记录相结合的方法来分析这些细胞类型在不同状态(饥饿与饱足)或特定任务(记忆测试、积极进食等)下的激发模式。 工具鼠标：MC4R-T2A-CRE Lepr-res-Cre，PACAP-Ires-Cre 病毒：AAV-flex-ChR2-mCherry，AAV-flex-synapsin-mCherry，AAV-flex-eNpHR-YFP，AAV-flex-hM3Dq-mCherry，AAV-flex-hM4Di-mCherry，改良狂犬病病毒 设备：用于测量食物摄入量的表型笼。用于活体录制的Plexon Ominplex。锐片麻花钻机。

The first experiment would be to use immunohistochemistry to investigate the overlapping/non-overlapping expression of Mc4rs and Leprs in the hippocampus. Both sets of neurons in the hypothalamus encode satiety signals and act to blunt feeding, of course with different signaling cascades and circuit mechanisms. Although neither of these receptors have good antibodies and in situs are nearly impossible given the low levels of expression, we can co-localized GFP-signaling from the Mc4r promoter-driven Cre line with STAT3 expression following leptin injection (see Myers et al 2012). Next, the obvious experiment is to transduce Lepr+ and/or Mc4r+ hippocampal neurons with ChR2 or eNpHR and assess the effects of photostimulation of photoinhibition on feeding (quantity as well as meal patterns including meal size, meal frequency, meal length) and body weight. If we see some convincing alteration in behavior, we are then in a great position again to dissect the circuit with regard to inputs and outputs using viral tools. Even more interesting is the ability to perform conditioned place preference and other memory/foraging tasks aimed at procuring food during optogenetic manipulation. We can perform these using a simple chamber or even design automated protocols using the specialized Phenotyper cages. As with the other projects listed above, we can (and definitely should) use a combination of in vitro and ex vivo slice recordings to analyze the firing patterns of these cell types in differential states (hungry versus sated) or specific tasks (memory assays, active eating ect). Tools Mice: Mc4r-t2a-Cre LepR-ires-Cre, Pacap-ires-Cre Virus: AAV-FLEX-ChR2-mCherry, AAV-FLEX-synapsin-mCherry, AAV-FLEX-eNpHR-YFP, AAV-FLEX-hM3Dq-mCherry, AAV-FLEX-hM4Di-mCherry, modified rabies virus Equipment: Phenotyper cage for measurements of food intake. Plexon Ominplex for in vivo recordings. Acute slice ephys rig.