Presynaptic Release of Endocannabinoids in the Nucleus Accumbens

描述(由申请人提供)：内源性大麻素(ECB)调节中枢神经系统中最普遍和最复杂的信号系统之一。它们是无处不在的兴奋性和抑制性神经传递的调节者，过去20年的研究已经很好地建立了ECB信号的逆行机制。具体地说，突触后神经元释放ECB，激活突触前定位的ECB受体1型(CB1R)，并调节神经递质的释放。这种逆行机制已经成为描述ECB如何调节突触传递的理论的中心原则，它们在许多大脑区域的兴奋性和抑制性突触上都是这样做的。然而，我们最近从腹侧被盖区(VTA)到伏隔核(NAC)的多巴胺能投射中获得了数据，这些数据描绘了与这一主导概念截然不同的情景。利用光遗传学、电子显微镜和电生理工具，我们的初步研究证明了一种形式的短期突触可塑性，这种可塑性可能是由多巴胺能轴突终末释放的ECB触发的。简而言之，i)选择性激活VTA到NAC的多巴胺能轴突触发对相邻突触的GABA传递的短期抑制；ii)GABA能突触的单独激活并不能触发这种效应；iii)抑制CB1Rs可以阻止这种异突触的可塑性；iv)在BAPTA对突触后的钙离子进行螯合并在强力性和代谢性谷氨酸受体阻滞剂的存在下，这种可塑性仍然存在；v)ANANDAME(AEA)是CNS中两个主要的ECB之一，N-酰基磷脂酰胺磷脂酶D(NAPE-PLD)是合成AEA的酶，在NAC内的突触前终末中被发现。这些结果提出了一种明显的可能性，即ECB可能是从多巴胺能突触前终末合成和释放的，并以交叉突触的方式调节相邻的GABA能突触。这种由ECB介导的细胞行为的新形式，如果得到证实，代表着首次展示了ECB在NAC的突触前释放。这个CEBRA R21应用程序将更全面地描述这一令人兴奋的新形式的ECB发布。我们将通过明确的方法，包括光遗传学、药理学、分子和电子显微镜分析，通过彻底表征ECB从VTA到NAC的多巴胺能投射来实现这一目标。具体来说，我们将确定介导异突触调节的ECB的类型(S)，产生ECB的合成酶(S)，以及ECB被释放的突触前或突触后部位。预期的结果将阐明突触前多巴胺能终末是否确实在NAC内释放ECB，从而可能为理解ECB系统的细胞和行为角色提供新的方向。鉴于ECB与几种大脑疾病的病理生理学有关，包括药物成瘾、抑郁症和肥胖症，这一系列研究是 与美国国立卫生研究院的任务高度相关。

DESCRIPTION (provided by applicant): Endocannabinoids (eCBs) mediate one of the most prevalent and complex signaling systems in the central nervous system. They are ubiquitous regulators of excitatory and inhibitory neural transmission, and studies over the past two decades have well established a retrograde mechanism of eCB signaling. Specifically, postsynaptic neurons release eCBs, which activate presynaptically located eCB receptor type 1 (CB1R) and regulate release of neurotransmitters. This retrograde mechanism has become the central tenet of theories describing how eCBs regulate synaptic transmission, which they do at both excitatory and inhibitory synapses in many brain regions. However, we have recently acquired data from dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) that depict a scenario in distinct contrast to this dominant concept. Using optogenetic, electron microscopic and electrophysiological tools, our preliminary studies demonstrate a form of short-term synaptic plasticity that is likely triggered by eCBs released from dopaminergic axon terminals. Briefly, i) Selective activation of VTA-to-NAc dopaminergic axons triggers short- term inhibition of GABA transmission at adjacent synapses; ii) Activation of GABAergic synapses alone does not trigger this effect; iii) Inhibiting CB1Rs prevents this heterosynaptic plasticity; iv) The plasticity persists following postsynaptic chelation of Ca2+ by BAPTA, and in the presence of inotropic and metabotropic glutamate receptor blockers; v) Anandamide (AEA) is one of the two major eCBs in the CNS, and N- acylphosphatidylethanolamine phospholipase D (NAPE-PLD), an enzyme that synthesizes AEA, is identified in presynaptic terminals within the NAc. These results raise a striking possibility tat eCBs may be synthesized and released from dopaminergic presynaptic terminals and regulate adjacent GABAergic synapses in a cross- synaptic manner. This novel form of eCB-mediated cellular behavior, if verified, represents the first demonstration of presynaptic release of eCBs i the NAc. This CEBRA R21 application will more thoroughly characterize this exciting new form of eCB release. We will achieve this goal by thoroughly characterizing eCB release from VTA-to-NAc dopaminergic projections with definitive approaches, including optogenetic, pharmacological, molecular, and electron microscopic assays. Specifically, we will determine the type(s) of eCB that mediates the heterosynaptic regulation, the synthetic enzyme(s) that produces the eCB, and the pre- or postsynaptic sites where the eCB is released. The expected results will clarify whether presynaptic dopaminergic terminals do indeed release eCBs within the NAc, and thus potentially provide a new direction for understanding the cellular and behavioral roles of the eCB system. Given that eCBs are implicated in the pathophysiology of several brain diseases, including drug addiction, depression, and obesity, this line of research is highly relevant to the mission of the NIH.