Correspondence of fentanyl brain pharmacokinetics and behavior measured via engineering opioids biosensors and computational ethology

Despite the ongoing epidemic of opioid use disorder and death by fentanyl overdose, opioids remain the gold standard for analgesics. Pharmacokinetics (PK) dictates the individual's experience and utility of drugs; however, PK and behavioral outcomes have been conventionally studied in separate groups, even in preclinical models. To bridge this gap, we developed the first class of sensitive, selective, and genetically encodable fluorescent opioid biosensors, iOpioidSnFRs, including the fentanyl sensor, iFentanylSnFR. We expressed iFentanylSnFR in the ventral tegmental area of mice and recorded [fentanyl] alongside videos of behaviors before and after administration. We developed a machine vision routine to quantify the effects of the behavior on locomotor activity. We found that mice receiving fentanyl exhibited a repetitive locomotor pattern that paralleled the [fentanyl] time course. In a separate experiment, mice navigating a complex maze for water showed a dosedependent impairment in navigation, in which animals repeated incorrect paths to the exclusion of most of the unexplored maze for the duration of the average fentanyl time course. This approach complements classical operant conditioning experiments and introduces a key feature of human addiction, the ability to carry out an ethologically relevant survival task, only now quantified in rodents. Finally, we demonstrate the utility of iFentanylSnFR in detecting fentanyl spiked into human biofluids and the generalizability of engineering methods to evolve selective biosensors of other opioids, such as tapentadol and levorphanol. These results encourage diagnostic and continuous monitoring approaches to personalizing opioid regimens for humans.