Department of Anesthesiology and Critical Care, University of Pennsylvania Perelman School of Medicine, Philadelphia, Philadelphia 19104.
Neuroscience of Unconsciousness and Reanimation Research Alliance, University of Pennsylvania, Perelman School of Medicine, Philadelphia, Philadelphia 19104.
J Neurosci. 2023 Mar 29;43(13):2338-2348. doi: 10.1523/JNEUROSCI.1884-22.2023. Epub 2023 Feb 27.
Photoaffinity ligands are best known as tools used to identify the specific binding sites of drugs to their molecular targets. However, photoaffinity ligands have the potential to further define critical neuroanatomic targets of drug action. In the brains of WT male mice, we demonstrate the feasibility of using photoaffinity ligands to prolong anesthesia via targeted yet spatially restricted photoadduction of azi--propofol (aziPm), a photoreactive analog of the general anesthetic propofol. Systemic administration of aziPm with bilateral near-ultraviolet photoadduction in the rostral pons, at the border of the parabrachial nucleus and locus coeruleus, produced a 20-fold increase in the duration of sedative and hypnotic effects compared with control mice without UV illumination. Photoadduction that missed the parabrachial-coerulean complex also failed to extend the sedative or hypnotic actions of aziPm and was indistinguishable from nonadducted controls. Paralleling the prolonged behavioral and EEG consequences of on target photoadduction, we conducted electrophysiologic recordings in rostral pontine brain slices. Using neurons within the locus coeruleus to further highlight the cellular consequences of irreversible aziPm binding, we demonstrate transient slowing of spontaneous action potentials with a brief bath application of aziPm that becomes irreversible on photoadduction. Together, these findings suggest that photochemistry-based strategies are a viable new approach for probing CNS physiology and pathophysiology. Photoaffinity ligands are drugs capable of light-induced irreversible binding, which have unexploited potential to identify the neuroanatomic sites of drug action. We systemically administer a centrally acting anesthetic photoaffinity ligand in mice, conduct localized photoillumination within the brain to covalently adduct the drug at its sites of action, and successfully enrich irreversible drug binding within a restricted 250 µm radius. When photoadduction encompassed the pontine parabrachial-coerulean complex, anesthetic sedation and hypnosis was prolonged 20-fold, thus illustrating the power of photochemistry to help unravel neuronal mechanisms of drug action.
光亲和配体是众所周知的工具，用于鉴定药物与其分子靶标的特定结合位点。然而，光亲和配体有可能进一步确定药物作用的关键神经解剖靶点。在 WT 雄性小鼠的大脑中，我们证明了使用光亲和配体通过靶向但空间受限的 azi--丙泊酚（aziPm）光加成作用来延长麻醉的可行性，aziPm 是全身麻醉丙泊酚的光反应类似物。在桥脑前头部用双侧近紫外光加成作用进行 aziPm 的全身给药，在脑桥臂旁核和蓝斑核的边界处，与没有 UV 照射的对照小鼠相比，镇静和催眠作用的持续时间增加了 20 倍。错过脑桥臂旁-蓝斑核复合体的光加成作用也未能延长 aziPm 的镇静或催眠作用，与未加成对照物无法区分。与靶标光加成作用的延长行为和 EEG 后果平行，我们在脑桥前脑切片中进行了电生理记录。使用蓝斑核内的神经元进一步突出不可逆 aziPm 结合的细胞后果，我们证明了在 brief bath application 时，不可逆的 aziPm 结合会导致自发动作电位短暂减慢，这种作用在光加成作用后变得不可逆。总之，这些发现表明基于光化学的策略是一种可行的新方法，可用于探测中枢神经系统的生理学和病理生理学。光亲和配体是能够进行光诱导不可逆结合的药物，具有未被开发的潜力来鉴定药物作用的神经解剖部位。我们在小鼠中系统地给予一种中枢作用的麻醉光亲和配体，在大脑内进行局部光照射，使药物在其作用部位共价加成，并且成功地在 250 µm 的限制半径内富集不可逆药物结合。当光加成作用包括桥脑臂旁-蓝斑核复合体时，麻醉镇静和催眠作用延长了 20 倍，从而说明了光化学的力量可以帮助揭示药物作用的神经元机制。
