Development of opioid and ketamine probes for in vivo photopharmacology

项目摘要 药理探针被广泛用于研究神经系统。尽管传统的小分子药物往往对靶受体表现出极高的特异性，但由于扩散，其作用缓慢且空间不精确。这阻碍了体内的神经药理学研究，特别是那些涉及高分辨率电生理、成像和行为跟踪方法的研究。这样的研究极大地受益于将测量与定义明确的、时间锁定的刺激相关联的能力，这些刺激的强度和持续时间可以很容易地变化。为了满足这一需求，我们正在开发可以系统地以不活跃的形式使用并随后使用短光闪光以高空间和时间精度在大脑中释放的“笼子”药物。为了与使用荧光探针的神经功能的光学测量兼容，我们将开发新的光化学保护基，这种保护基的波长可调为与常见的绿色和红色荧光团在单光子和双光子激发方面的光谱正交。我们将进一步优化这些“笼子”群，以促进由此产生的笼子药物的脑部穿透。我们将通过将新的笼状基团纳入第二代笼状阿片类药物来评估它们的实用性，这些笼状阿片类药物应该比我们的第一代变种具有显著的实验优势，第一代变种只能用紫外光激活。我们将使用体外、体外和体内实验范式严格验证新的笼式阿片类药物，最终完成阿片类药物诱发的神经化学信号的行为分析和纤维光度记录。此外，我们将开发笼式氯胺酮衍生物，可用于研究解离状态下的神经机制，以及氯胺酮的快速抗抑郁作用。笼养的氯胺酮变种也将使用体外、体外和体内实验范式进行评估，包括测量前额叶皮质的棘突发生和神经活动。这些努力将包括开发一种光学配置，用于同时进行双光子成像和通过植入的棱镜进行单光子光解。为了最大限度地利用终端用户，这两个笼式药物家族的性能标准都是通过与科学界的广泛协商来确定的。

Project Summary Pharmacological probes are widely used to study the nervous system. Despite often exhibiting exquisite specificity for target receptors, due to diffusion, traditional small molecule drugs act slowly and with spatial imprecision. This impedes neuropharmacological studies in vivo, particularly those involving high-resolution electrophysiological, imaging, and behavioral tracking methods. Such studies greatly benefit from the ability to correlate measurements with well-defined, time-locked stimuli that can be readily varied in intensity and duration. To meet this need, we are developing “caged” drugs that can be applied systemically in an inactive form and subsequently released in the brain with high spatial and temporal precision using short light flashes. To enable compatibility with optical measurements of neural function involving fluorescent probes, we will develop new photochemical protecting groups that are wavelength tuned to be spectrally orthogonal to common green and red fluorophores, in terms of both one-photon and two-photon excitation. We will further optimize these “caging” groups to facilitate the brain penetrance of the resulting caged drugs. We will evaluate the utility of new caging groups by incorporating them into 2nd generation caged opioid drugs that should provide significant experimental advantages over our 1st generation variants, which could only be photoactivated with ultraviolet light. We will rigorously validate new caged opioid drugs using in vitro, ex vivo, and in vivo experimental paradigms, culminating in behavioral assays and fiber photometry recordings of opioid-evoked neurochemical signaling. In addition, we will develop caged ketamine derivatives that can be used to study the neural mechanisms underlying dissociative states, as well as ketamine’s rapid antidepressant actions. Caged ketamine variants will also be evaluated using in vitro, ex vivo, and in vivo experimental paradigms, including measurements of spinogenesis and neural activity in the prefrontal cortex. These efforts will involve the development of an optical configuration for simultaneous two-photon imaging and one photon photolysis through implanted prisms. To maximize end-user uptake, performance criteria for both caged drug families are determined through extensive consultation with the scientific community.