In Vivo Photoadduction of Anesthetic Ligands in Mouse Brain Markedly Extends Sedation and Hypnosis.

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 in vivo to prolong anesthesia via targeted yet spatially restricted photoadduction of azi-m-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 in vivo 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.SIGNIFICANCE STATEMENT 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 in vivo 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 in vivo photochemistry to help unravel neuronal mechanisms of drug action.

A high-throughput approach for identification of novel general anesthetics.

Anesthetic development has been a largely empirical process. Recently, we described a GABAergic mimetic model system for anesthetic binding, based on apoferritin and an environment-sensitive fluorescent probe. Here, a competition assay based on 1-aminoanthracene and apoferritin has been taken to a high throughput screening level, and validated using the LOPAC(1280) library of drug-like compounds. A raw hit rate of approximately 15% was reduced through the use of computational filters to yield an overall hit rate of approximately 1%. These hits were validated using isothermal titration calorimetry. The success of this initial screen and computational triage provides feasibility to undergo a large scale campaign to discover novel general anesthetics.

Risk factors for venous gas emboli after decompression from prolonged hyperbaric exposures.

INTRODUCTION: The physical forces governing gas phase nucleation and growth in a liquid would predict less variation in the development of decompression sickness (DCS) than is known to occur in people. METHODS: In order to gain insight into the causes of biological susceptibility to DCS, we analyzed a dataset containing 250 human steady-state hyperbaric exposures using multivariate ordinal and linear regression analysis for relationships between venous gas emboli (VGE) and exposure parameters and subject characteristics. RESULTS: In both previously published data and new chamber exposure data, we found that the strongest predictor of VGE magnitude after decompression was the duration and depth of the hyperbaric exposure, as predicted. Of the subject factors, only age was significantly associated with VGE; body mass index (BMI) and gender were not. The relationship between age and VGE strengthened with decompression magnitude. DISCUSSION: These results suggest that the physiology of aging interacts with the mechanism of VGE generation, altering the risk of DCS after decompression.