Corticostriatal mechanisms of action learning and habit formation

纹状体黑质直接通路2-花生四烯酸甘油参与乙醇对突触传递和行为的影响 内源性大麻素是激活大麻素受体的脂质代谢产物，这些受体是大麻药物中的主要精神活性成分--三角洲-9-四氢大麻酚的主要靶标。CB1受体的内源性大麻素激活抑制了大脑、脊髓和周围神经系统突触处神经递质的释放。这种受体的激活与控制酒精摄入量和其他与酒精有关的行为有关。然而，在这些行为中，内源性大麻素信号在特定脑区和特定突触中的作用尚不完全清楚。纹状体是参与动作学习和控制的大脑皮质下区域，表达高水平的内源性大麻素和CB1受体。背外侧纹状体(DLS)是前脑感觉运动回路的一部分，与过量饮酒有关。因此，我们假设酒精改变了DLS中的内源性大麻素信号，对这个和其他纹状体亚区的影响有助于酒精的摄入。 为此，我们首先测量了含有DLS的脑片中的内源性大麻素信号。为了测量DLS神经元皮质传入的内源性大麻素信号，我们通过体内病毒注射在小鼠初级运动皮质(M1)表达了基于G蛋白偶联受体激活的基因编码的内源性大麻素感受器(GRABeCB2.0)。GRABeCB2.0传感器随后被传输到DLS的皮质终端。我们制备了脑片，并用光度记录的方法检测了电刺激DLS所激活的荧光信号。与我们之前的研究一样，我们发现，短时间的电刺激会产生持续10秒的荧光增强。这种增加在不结合内源性大麻素的突变体Grab结构中没有观察到。一种阻止内源性大麻素与GRABeCB2.0结合的药物和一种阻断二酰基甘油脂肪酶阿尔法(DGLpha)的药物也阻止了荧光增加，DGLpha是催化内源性大麻素2-花生单酰甘油(2-AG)产生的酶。这些发现与我们小组以前的研究一致，表明2-AG是这种电刺激模式下释放的主要内源性大麻素。接下来，我们使用遗传方法来确定DLS中的哪些神经元产生我们正在检测的2-AG。在之前的一项研究中，我们发现从所有纹状体棘突投射神经元(SPN)中移除DGLpha的基因消除了GRABeCB2.0测量的刺激驱动的2-AG释放。在目前的研究中，我们仅从投射到黑质网状部的SPN(“直接”通路SPN)中专门去除了DGLα。有些令人惊讶的是，我们发现这种去除强烈地减少了GRABeCB2.0测量的2-AG的释放。这表明，SPN的直接通路对刺激诱导的纹状体2-AG释放的贡献比预期的更大。 然后，我们研究了急性酒精应用对M1皮质终末表达GRABeCB2.0的脑片中2-AG释放的影响。当药物存在于切片中时，酒精抑制了刺激诱导的2-AG的释放，当酒精从切片中移除时，这种作用被逆转。我们还发现，这种急性酒精暴露抑制了从直接途径SPN中去除DGLpha后残留的少量2-AG的释放。因此，酒精似乎可以减少所有SPN亚型的2-AG释放。 接下来，我们研究了直接途径SPN产生2-AG在酒精相关行为中的潜在作用，其理论基础是纹状体中这种内源性大麻素的大部分释放来自这些神经元。我们再次使用了DGLpha基因从直接途径SPN中移除的小鼠。我们观察到，在缺乏直接通路SPN DGLpha的小鼠中，酒精导致的翻正反射丧失的持续时间缩短了。这表明SPN亚型释放的2-AG参与了酒精的镇静作用。我们还在直接途径SPN DGLalpa基因敲除小鼠中进行了两瓶选择饮酒的实验，观察到雄性小鼠的酒精偏好降低，水的偏好增加，但雌性小鼠没有。这些小鼠对甜味和苦味物质的偏好没有改变。因此，直接通路SPN释放的2-AG和纹状体内的内源性大麻素信号似乎在控制饮酒中起着特殊的作用。 在未来的实验中，确定纹状体内大麻素信号的哪些细胞靶点对酒精的影响将是有趣的。同样重要的是要确定纹状体内源性大麻素和CB1信号的其他操作，例如抑制2-AG的降解，是否会改变酒精相关的行为。

Striatonigral direct pathway 2-arachidonoylglycerol contributes to ethanol effects on synaptic transmission and behavior Endocannabinoids are lipid metabolites that activate cannabinoid receptors, the same receptors that are the major targets for delta-9-tetrahydrocannabinol, the major psychoactive ingredient in cannabis drugs. Endocannabinoid activation of the CB1 cannabinoid receptor inhibits release of neurotransmitters at synapses throughout the brain, spinal cord and peripheral nervous system. Activation of this receptor has been implicated in the control of alcohol intake and other alcohol-related behaviors. However, the roles in these behaviors of endocannabinoid signaling in specific brain regions and at specific synapses are not fully understood. The striatum, a subcortical brain region involved in action learning and control, expresses high levels of endocannabinoids and CB1 receptors. The dorsolateral striatum (DLS), a part of the forebrain sensorimotor circuitry, has been implicated in excessive alcohol intake. Thus, we hypothesized that alcohol alters endocannabinoid signaling in DLS and effects on this and other striatal subregions contribute to alcohol intake. To this end, we began by measuring endocannabinoid signaling in brain slices containing DLS. To measure endocannabinoid signaling at cortical inputs to DLS neurons we expressed the genetically-encoded fluorescent G protein-coupled receptor activation-based endocannabinoid sensor (GRABeCB2.0) in mouse primary motor cortex (M1) by in vivo viral injection. The GRABeCB2.0 sensor was then trafficked to cortical terminals in DLS. We prepared brain slices and used photometric recording to examine the fluorescent signals activated by electrical stimulation in DLS. As in our previous studies, we found that short bursts of electrical stimuli produced an increase in fluorescence that persisted for 10s of seconds. This increase was not observed with a mutant GRAB construct that does not bind endocannabinoids. The fluorescence increase was also prevented by a drug that blocks endocannabinoid binding to GRABeCB2.0 and a drug that blocks diacylglycerol lipase alpha (DGLalpha) the enzyme that catalyzes production of the endocannabinoid 2-arachidonoyl glycerol (2-AG). These findings are consistent with previous studies in our group indicating that 2-AG is the main endocannabinoid released following this pattern of electrical stimulation. We next used a genetic approach to determine which neurons in DLS produce the 2-AG we are detecting. In a previous study, we found that genetic removal of DGLalpha from all striatal spiny projection neurons (SPNs) eliminates stimulus-driven 2-AG release measured with GRABeCB2.0. In the persent study we specifically removed DGLalpha from only those SPNs that project to the substantia nigra pars reticulata (the "direct" pathway SPNs). Somewhat surprisingly, we found that this removal strongly reduced 2-AG release as measured with GRABeCB2.0. This indicates that the direct pathway SPNs contribute more strongly than expected to stimulation-induced 2-AG release in striatum. We then examined the effects of acute alcohol application on 2-AG release in brain slices expressing GRABeCB2.0 in M1 cortical terminals. Alcohol inhibited the stimulus-induced 2-AG release while the drug was present in the slice, and this effect was reversed when alcohol was removed from the slice. We also found that this acute alcohol exposure inhibited the small amount of 2-AG release that remained after DGLalpha removal from direct pathway SPNs. Thus, alcohol appears to reduce 2-AG release from all SPN subtypes. We next examined the potential role of 2-AG production from direct pathway SPNs in alcohol-related behaviors, with the rationale that the majority of release of this endocannabinoid in striatum comes from these neurons. We once again used mice in which DGLalpha was genetically removed from the direct pathway SPNs. We observed that the duration of alcohol-induced loss of righting reflex was reduced in the mice lacking direct pathway SPN DGLalpha. This suggests that 2-AG release from this SPN subtype contributes to the sedative effect of alcohol. We also performed a two-bottle choice alcohol drinking paradigm in the direct pathway SPN DGLalpa knockout mice and observed decreased alcohol preference and increased water preference in male, but not female mice. Changes in preference for sweet and bitter tasting substances was not altered these mice. Thus, 2-AG release from direct pathway SPNs and endocannabinoid signaling in striatum appear to have specific roles in control of alcohol drinking. In future experiments it will be interesting to determine what cellular targets of striatal endocannabinoid signaling contribute to the effects of alcohol. It will also be important to determine if other manipulations of striatal endocannabinoid and CB1 signaling, e.g. inhibition of 2-AG degradation, alter alcohol-related behaviors.