Investigating addictive/compulsive feeding behavior

确定长期激活AGRP或POMC对成瘾或强迫进食行为的影响。假设：AGRPARC的慢性激活会导致强迫摄食行为，而POMCARC的慢性激活会导致慢性摄食行为的减少。 理论基础：研究表明，AGRP神经元的急性光遗传激活在一小时内驱动摄食行为(Aponte等人，2011年)。虽然慢性光刺激还没有报道，但DREADD介导的5天AGRP神经元的激活已经被证明促进了由于过度吞噬而导致的体重增加和脂肪质量(Krash等人，2011年)。此外，POMC神经元的慢性24小时激活由于吞噬减少而导致显著的体重减轻(Aponte等人，2011年)。利用现有的光遗传学工具和表达Cre基因的转基因动物，我们将评估AGRPARC或POMCARC神经元长期慢性激活后的永久性行为变化。 方法：在AGRP-ILES-CRE和POMC-CRE小鼠品系的下丘脑弓状体内注射ChR2。在3周的转导期后，动物将在一个月的过程中接受AGRPARC或POMCARC神经元的慢性刺激(每隔一小时进行一次光刺激；每天12小时)。在这个刺激月期间，将监测和控制食物摄入量，以确保与对照仔猪相匹配的每日食物摄入量。这是势在必行的，因为AGRP刺激的小鼠将持续摄入更多的食物，而POMC刺激的小鼠将持续比产仔对照组小鼠摄入更少的食物，而体重需要匹配以保证准确性，因为肥胖小鼠吃得更多，瘦小鼠消耗更少的食物。在一个月的刺激方案之后，动物将被给予随意的食物访问，同时测量喂养参数(食物消耗量、进食持续时间、喂养回合)以及运动活动和觅食行为，并与对照组进行比较。此外，这些动物将被评估它们是否愿意为食物而工作，使用上面描述的戳鼻子试验。 预测、解释和未来实验：我们预计这些实验不会有任何问题，因为考虑到我们可以接触到必要的小鼠、病毒试剂和设备，这些研究的执行非常简单。我们预测，促进饥饿的AGRP回路的慢性激活将导致与摄食相关的行为的永久性增加，而促进饱腹感的POMC回路的慢性激活将导致与摄食相关的行为的永久性减少。如果像我们预测的那样，我们检测到摄食行为的变化，我们将进行额外的实验来评估两个下游信号通路(例如，同时刺激或抑制MC4RPVN神经元以分别阻断慢性AGRP或POMC激活的影响)。如果我们得到否定的结果，并且在长达一个月的刺激后饮食行为没有改变，我们可以观察直接的慢性刺激或抑制位于AGRPARC和POMCARC神经元下游的神经元的效果。

To determine the impact of chronic activation of AGRP or POMC on addictive or compulsive feeding behavior. Hypothesis: Chronic activation of AGRPARC will result in compulsive feeding behavior, and chronic POMCARC activation will result in chronically reduced feeding behavior. Rationale: Studies have demonstrated that acute optogenetic-activation of AGRP neurons drives feeding behavior over the course of an hour (Aponte et al., 2011). While chronic photo-stimulation has not been reported, a 5-day DREADD-mediated activation of AGRP neurons has been shown to promote weight gain and fat mass due to hyperphagia (Krashes et al., 2011). Furthermore, chronic 24 hr activation of POMC neurons led to significant weight loss due to hypophagia (Aponte et al., 2011). Using the available optogenetic tools and transgenic Cre-expressing animals, we will assess the permanent behavior changes after chronic activation of AGRPARC or POMCARC neurons over a prolonged period of time. Approach: Both AGRP-ires-Cre and POMC-Cre mouse lines will be bilaterally injected with ChR2 in the hypothalamic ARC. After a 3-week transduction period, the animals will be subjected to chronic stimulation of either AGRPARC or POMCARC neurons over the course of a month (photostimulation every other hour; 12 hours a day). Food intake will be monitored and controlled for the duration of this stimulation month to ensure matching daily food intake as the control littermates. This is imperative as AGRP-stimulated mice will continually eat more food and POMC-stimulated mice will continually eat less food than littermate controls and body weights need to be matched for accuracy, as obese mice eat more and lean mice consume less food. Following a month of the stimulation protocol, animals will be given ad libitum food access while feeding parameters (quantity of food consumed, duration of meals, feeding bouts), as well as locomotor activity and foraging behavior are measured and compared to controls. Additionally, these animals will be assessed for their willingness to work for food using a nose poke assay described above. Predictions, interpretations & Future Experiments: We do not anticipate any problems with these experiments, as these studies are very straightforward to perform given our access to the necessary mice, viral reagents and equipment. We predict that chronic activation of hunger-promoting AGRP circuitry will lead to permanent increases in feeding-related behaviors, while chronic activation of satiety-promoting POMC circuitry will lead to permanent decreases in feeding-related behaviors. If as we predict, we detect changes in feeding behavior, we will perform additional experiments to assess both the downstream signaling pathways (for example, simultaneously stimulate or inhibit MC4RPVN neurons to occlude the effects of chronic AGRP or POMC activation, respectively). If we obtain negative results and eating behavior is not altered following a month-long stimulation, we can look at the effects of either direct chronic stimulation or inhibition of the neurons identified as sitting downstream from AGRPARC and POMCARC neurons.