Synaptic Transmission: Modulation, Plasticity And Effects Of Drugs Of Abuse

纹状体内源性大麻素信号转导的动力学 内源性大麻素（eCB）是含花生四烯酸的脂质的脂肪酸代谢物，其作用于大麻素1型和2型（CB 1和CB 2）受体以调节神经元功能。在纹状体中，eCBs参与了短期和长期突触抑制，并且eCBs还调节苍白球和实质性nivea中纹状体投射神经元的突触传递。然而，驱动eCB释放的因素、释放的时间和eCB信号的终止已经从间接测量中推断出来。为了直接测量背侧纹状体和苍白球脑切片中的eCB动力学，我们将荧光遗传编码的G蛋白偶联受体（GPCR）激活的内源性大麻素传感器（GRABeCB，由北京大学的Ao Dong和Yulong Li博士开发的修饰的CB 1受体）的表达与切片荧光测定法/光度法相结合。我们通过AAV注射在纹状体神经元或初级运动皮层（M1）神经元中表达GRABeCB，然后收集背侧纹状体和苍白球的荧光信号。使用了两个GRABeCB版本1.0和2.0。短暂的电刺激（例如，5脉冲在20 Hz）引起荧光增加是否测量从纹状体神经元，突触前M1传入投射到纹状体或纹状体间接通路投射到苍白球。荧光增加的开始发生在刺激开始后100毫秒，并且在刺激停止后持续增加10-15秒。 刺激爆发诱导的荧光增加被TTX或低细胞外钙阻断，也被阻止应用CB 1拮抗剂AM 251。因此，信号来自神经元。此外，当表达具有降低的eCB亲和力的F177 A突变体GRAB-eCB构建体时，不能诱发增加。刺激爆发诱导的荧光增加的幅度和持续时间增强的单酰基甘油脂肪酶，催化分解的eCB 2-花生四烯酸（2-AG）的酶的阻滞剂。 mGlu 5负变构调节剂MPEP和毒蕈碱乙酰胆碱受体1型拮抗剂减少刺激爆发诱导的纹状体eCB增加，与这些受体在eCB产生中的作用一致。eCB的增加也受到D2型多巴胺受体激活的抑制。我们目前正在研究刺激对纹状体突触可塑性的影响。我们的研究结果表明，GRABeCB是一个有用的工具，实时测量脑组织中的eCB水平。 我们目前还在研究不同纹状体神经元亚型中2-AG合成酶二酰甘油脂肪酶α（DGL α）在纹状体eCB生产中的作用。此外，我们正在研究乙醇对eCB产生/释放的影响，初步数据表明乙醇抑制eCB信号传导。未来的研究将检查慢性乙醇消耗和暴露对eCB信号传导的影响以及eCB产生和释放的机制。还将检查eCB信号变化对睡眠和酒精相关行为的影响。 纹状体神经元的cAMP信号转导 几种神经调节剂，包括乙酰胆碱，多巴胺和上述内源性大麻素激活GPCR，改变细胞内第二信使环AMP（cAMP）的水平。cAMP水平的变化反过来改变突触传递、突触结构和神经元基因表达。这些变化中的许多涉及cAMP依赖性蛋白激酶（PKA）的cAMP激活。为了更好地理解神经调节剂如何在真实的时间内响应于突触激活而改变细胞内cAMP和PKA水平，我们已经开始使用遗传编码的荧光传感器来使用光度法和细胞内成像方法检测脑切片中的cAMP和PKA磷酸化/激活。我们正在研究激活不同传入输入对纹状体直接通路和间接通路投射神经元中cAMP相关细胞内信号传导的影响，以确定这些神经元中GPCR的不同互补物如何影响这种信号传导，以及如何有助于突触调制和突触可塑性。迄今为止的研究结果表明，传入刺激纹状体切片产生的直接通路投射神经元，介导的D1多巴胺受体，耦合到GS型G蛋白的cAMP的大幅增加。这些增加持续10秒后，单一传入刺激，持久的增加多巴胺本身（如快速扫描循环伏安法测量）。目前，我们正在研究刺激诱导的cAMP的变化，在间接途径投射神经元，和其他受体的作用，cAMP控制神经元从两个途径。 乙醇也被认为可以改变cAMP的产生，这种药物的分子作用可能有助于对突触传递以及酒精相关行为的影响。因此，我们感兴趣的是确定急性和慢性乙醇是否会改变纹状体突触前和突触后元件中cAMP的产生和PKA的激活。这些发现将帮助我们确定在未来的研究中，在哪里进行旨在改变cAMP/PKA信号传导的操作，最终目的是改变酒精相关行为。

Dynamics of Endocannabinoid Signaling in Striatum Endocannabinoids (eCBs) are fatty acid metabolites of arachidonic acid-containing lipids that act on the cannabinoid type 1 and 2 (CB1 and CB2) receptors to modulate neuronal function. In the striatum, eCBs have been implicated in short- and long-term synaptic depression, and eCBs also modulate transmission at synapses made by striatal projection neurons in globus pallidus and substantial nivea. However the factors that drive eCB release, timing of release and termination of the eCB signal have been inferred from indirect measures. To directly measure eCB dynamics in dorsal striatal and globus pallidus brain slices we combined expression of the fluorescent genetically encoded G Protein-Coupled Receptor (GPCR) Activation-based Sensor for endocannabinoids (GRABeCB, a modified CB1 receptor developed by Drs. Ao Dong and Yulong Li at Peking University) with slice fluorimetry/photometry. We either expressed GRABeCB in striatal neurons or in primary motor cortex (M1) neurons by AAV injection, and then collected fluorescent signals in dorsal striatum and globus pallidus. Two GRABeCB versions, 1.0 and 2.0, were used. Brief bursts of electrical stimuli (e.g. 5 pulses at 20 Hz) elicited increases in fluorescence whether measurements were made from striatal neurons, presynaptic M1 afferents projecting into striatum or striatal indirect pathway projections into the globus pallidus. The onset of the fluorescence increases occurred 100s of msec after stimulation onset, and increases persisted for 10-15 sec after stimulus cessation. The stimulus burst-induced fluorescence increases were blocked by TTX or in low extracellular calcium and were also prevented by application of the CB1 antagonist AM251. Thus, signals were of neuronal origin. Furthermore, increases could not be evoked when the F177A mutant GRAB-eCB construct with reduced eCB affinity was expressed. The amplitude and duration of stimulus burst-induced fluorescence increases was enhanced by a blocker of monoacylglycerol lipase, the enzyme that catalyzes breakdown of the eCB 2-arachadonoylglycerol (2-AG). The mGlu5 negative allosteric modulator MPEP and antagonists of muscarinic acetylcholine receptor type 1 reduced stimulus burst-induced eCB increases in striatum, consistent with the proposed role for these receptor in eCB production. The eCB increases were also depressed by activation of D2-type dopamine receptors. We are currently examining effects of stimuli that produce striatal synaptic plasticity. Our findings to date indicate that GRABeCB is a useful tool for real-time measurement of eCB levels in brain tissue. We are also currently examining the role in striatal eCB production of the 2-AG synthesis enzyme diacylglycerol lipase alpha (DGLalpha) in different striatal neuronal subtypes. In addition, we are examining effects of ethanol on eCB production/release, and preliminary data indicate that ethanol inhibits eCB signaling. Future studies will examine effects of chronic ethanol consumption and exposure on eCB signaling and the mechanisms underlying eCB production and release. The consequences of the eCB signaling changes on sleep and alcohol-related behaviors will also be examined. Cyclic AMP Signaling in Striatal Neurons Several neuromodulators, including acetylcholine, dopamine and the aforementioned endocannabinoids activate GPCRs that alter intracellular levels of the second messenger cyclic AMP (cAMP). Changes in cAMP levels, in turn, alter synaptic transmission, synapse structure and neuronal gene expression. Many of these changes involve cAMP activation of the cAMP-dependent protein kinase (PKA). To better understand how neuromodulators alter intracellular cAMP and PKA levels in real time in response to synaptic activation, we have begun to use genetically-encoded fluorescent sensors to detect cAMP and PKA phosphorylation/activation in brain slices using photometry and intracellular imaging approaches. We are examining effects of activating different afferent inputs on cAMP-related intracellular signaling in the striatal direct pathway and indirect pathway projection neurons to determine how the different complements of GPCRs in these neurons affect this signaling and how that contributes to synaptic modulation and plasticity at synapses onto these neurons. Findings to date indicate that afferent stimulation in striatal slices produced large increases in cAMP in direct pathway projection neurons that are mediated by D1 dopamine receptors that couple to the Gs-type G protein. These increases persist for 10 sec following single afferent stimuli, outlasting the increase in dopamine itself (as measured by fast-scan cyclic voltammetry). We are currently examining stimulus-induced cAMP changes in indirect pathway projection neurons, and the role of other receptors in cAMP control in neurons from both pathways. Ethanol is also known to alter cAMP production, and this molecular action of the drug may contribute to effects on synaptic transmission as well as alcohol-related behaviors. We are thus interested in determining if acute and chronic ethanol alters cAMP production and PKA activation, in both pre- and postsynaptic elements in the striatum. These findings will help us determine where to target manipulations aimed at altering cAMP/PKA signaling in future studies, with the ultimate aim of altering alcohol-related behaviors.