Investigating addictive/compulsive feeding behavior

确定AGRP或POMC的慢性激活对成瘾或强迫进食行为的影响。假设：AGRPARC的慢性激活将导致强迫性进食行为，而POMCARC的慢性激活将导致进食行为的慢性减少。 基本原理：研究已经证明，AGRP神经元的急性光遗传学激活在一小时的过程中驱动进食行为（Aponte等人，2011年）。虽然尚未报道慢性光刺激，但已显示5天DREADD介导的AGRP神经元活化促进由于摄食过多引起的体重增加和脂肪量（Krashes等人，2011年）。此外，POMC神经元的慢性24小时活化导致由于摄食不足引起的显著体重减轻（Aponte等人，2011年）。使用可用的光遗传学工具和转基因表达Cre的动物，我们将评估AGRPARC或POMCARC神经元长期慢性激活后的永久行为变化。 方法：AGRP-ires-Cre和POMC-Cre小鼠系将在下丘脑ARC中双侧注射ChR 2。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（每隔一小时光刺激;每天12小时）。在该刺激月期间，将监测和控制摄食量，以确保每日摄食量与对照同窝仔相匹配。这是必要的，因为AGRP刺激的小鼠将持续吃更多的食物，而POMC刺激的小鼠将持续吃比同窝对照小鼠更少的食物，并且体重需要匹配以获得准确性，因为肥胖小鼠吃得更多而瘦小鼠消耗的食物更少。在刺激方案一个月后，将给予动物自由进食，同时测量进食参数（摄食量、进餐持续时间、进食回合）以及运动活动和觅食行为，并与对照组进行比较。此外，将使用上述鼻戳试验评估这些动物是否愿意为食物工作。 预测，解释和未来的实验：我们预计这些实验不会有任何问题，因为这些研究非常简单，因为我们可以获得必要的小鼠，病毒试剂和设备。我们预测，促进饥饿的AGRP回路的慢性激活将导致进食相关行为的永久性增加，而促进饱腹感的POMC回路的慢性激活将导致进食相关行为的永久性减少。如果正如我们预测的那样，我们检测到进食行为的变化，我们将进行额外的实验来评估下游信号通路（例如，同时刺激或抑制MC 4 RPVN神经元以分别阻断慢性AGRP或POMC激活的影响）。如果我们获得阴性结果，并且在长达一个月的刺激后饮食行为没有改变，我们可以观察直接慢性刺激或抑制位于AGRPARC和POMCARC神经元下游的神经元的影响。

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.