Characterization of the Neural Circuits Underlying the Rapid Antidepressant Effect of Ketamine

描述(申请人提供)：严重的抑郁障碍给社会带来了巨大的负担，估计有五分之一的美国人在他们的一生中受到影响，其显著症状是快感乏力、精力不足、注意力不集中以及睡眠和食欲失调。这种疾病的流行与缺乏快速有效的治疗形成了鲜明对比。药物疗法或行为疗法通常需要八周或更长时间才能缓解症状，多达一半的患者对最初的治疗没有反应。最近的研究表明，单一小剂量的氯胺酮--一种增加大脑中谷氨酸信号的NMDA拮抗剂--可以在几个小时内缓解症状，即使是对传统抗抑郁药物治疗无效的患者也是如此。氯胺酮的新奇抗抑郁机制尚未完全被了解，这方面的研究迫在眉睫，因为氯胺酮有副作用(包括分离症状)和滥用潜力，使其不适合广泛使用。初步研究表明，氯胺酮的抗抑郁作用可以通过刺激下缘前额叶皮质(IlPFC)中的谷氨酸能细胞来再现，该区域已被证明在给予氯胺酮后不久经历了谷氨酸释放的高峰。这一关键发现需要刺激的空间、时间和细胞类型的特异性，这是通过将病毒载体注射到ilPFC中来实现的，该病毒载体将仅在含有谷氨酸能细胞标志CaMKII的细胞中表达通道视紫红质2(ChR2)。然后，这些细胞被激光刺激，其频率和持续时间旨在模仿全身注射氯胺酮的时间过程，直接连接到固定在头骨上的光纤插管。目标1将研究这种效应背后的特定ilPFC轴突投射。重要的是，病毒载体驱动ChR2在整个细胞中表达，甚至在轴突的末端也是如此。为了了解ilPFC中哪些谷氨酸能细胞负责抗抑郁作用，插管将 被放置以将光引导到接受ilPFC终末投射的区域之一，该区域包括伏核(NAC)、外侧缰核(LHB)和中缝背核(DR)，最近的研究表明，每一个区域都参与了情绪调节和抗抑郁反应。目标2将描述这些区域内的ilPFC投影的影响。利用免疫组织化学技术，在刺激后不久，每个大脑区域的切片将被c-Fos抗体染色，这是神经活动的标志，以评估大脑区域是由于ilPFC的刺激而变得更活跃还是更不活跃。此外，组织将被 与CaMKII或GAD的抗体共染色，以评估ilPFC投射突触到哪些细胞类型。这个项目将增加对快速抗抑郁效果背后的电路的理解，以帮助指明广泛临床使用的安全的氯胺酮类药物疗法的方向。

DESCRIPTION (provided by applicant): Major depressive disorder imposes a large burden on society, afflicting an estimated one in five Americans over their lifetime with its hallmark symptoms of anhedonia, low energy, loss of concentration, and sleep and appetite dysregulation. The prevalence of the disease contrasts with the paucity of rapid, effective treatments. Drug therapies or behavioral therapy typically take eight weeks or more to bring symptom relief, and up to half of patients do not respond to initial treatment. Recent studies have shown that a single low dose of ketamine, an NMDA antagonist that increases glutamate signaling in the brain, can bring about symptom relief in just a few hours, even in patients who do not respond to traditional antidepressant therapies. Ketamine's novel antidepressant mechanism has yet to be fully understood, and there is a pressing need for research in this area because ketamine has side effects (including dissociative symptoms) and abuse potential that make it unsuitable for widespread use. Preliminary studies have shown that ketamine's antidepressant effect can be reproduced by stimulation of glutamatergic cells in the infralimbic prefrontal cortex (ilPFC), an area that has been shown to undergo a spike in glutamate release shortly after ketamine administration. This key finding requires spatial, temporal, and cell-type specificity of stimulation, which is attained by injecting a viral vector into the ilPFC that will express channelrhodopsin 2 (ChR2), a light-sensitive protein, only in cells containing CAMKII, a marker for glutamatergic cells. These cells are then stimulated by a laser, at a frequency and duration intended to mimic the time course of systemic ketamine administration, connected directly to a fiberoptic cannula secured to the skull. Aim 1 will investigate the specific ilPFC axonal projections that underlie this effect. Importantly, the viral vector drives expression of ChR2 throughout the cell, even in the distal terminals of axons. To understand which glutamatergic cells within ilPFC are responsible for the antidepressant effect, the cannula will be placed to direct light to one of the regions that receives terminal projections from the ilPFC, which includes the nucleus accumbens (NAc), lateral habenula (lHb), and dorsal raphe (dR), each of which has been suggested by recent research to be involved in mood regulation and antidepressant response. Aim 2 will characterize the effect of the ilPFC projection within each of these areas. Using immunohistochemistry, slices of each brain area will be stained with a c-Fos antibody, a marker of neural activity, shortly after stimulation to assess if the brain region becomes more or less active as a result of stimulation from the ilPFC. Further, the tissue will be co-stained with antibodies for either CAMKII or GAD, a protein found in interneurons, to assess which cell types the ilPFC projections synapse onto. This project will increase understanding of circuits that underlie a rapid antidepressant effect to help point the way to ketamine-like pharmacotherapies that would be safe for widespread clinical use.