Proportional Downscaling of Glutamatergic Release Sites by the General Anesthetic Propofol atDrosophilaMotor Nerve Terminals

Abstract

Propofol is the most common general anesthetic used for surgery in humans, yet its complete mechanism of action remains elusive. In addition to potentiating inhibitory synapses in the brain, propofol also impairs excitatory neurotransmission. We use electrophysiological recordings from individual glutamatergic boutons in male and female larval Drosophila melanogaster motor nerve terminals to characterize this effect. We recorded from two bouton types, which have distinct presynaptic physiology and different average numbers of release sites or active zones. We show that a clinically relevant dose of propofol (3 mM) impairs neurotransmitter release similarly at both bouton types by decreasing the number of active release sites by half, without affecting release probability. In contrast, an analog of propofol has no effect on glutamate release. Coexpressing a truncated syntaxin1A protein in presynaptic boutons completely blocked this effect of propofol. Overexpressing wild-type syntaxin1A in boutons also conferred a level of resistance by increasing the number of active release sites to a physiological ceiling set by the number of active zones or T-bars, and in this way counteracting the effect of propofol. These results point to the presynaptic release machinery as a target for the general anesthetic. Proportionally equivalent effects of propofol on the number of active release sites across the different bouton types suggests that glutamatergic circuits that involve smaller boutons with fewer release sites may be more vulnerable to the presynaptic effects of the drug.

Synaptic Plasticity Induced by Differential Manipulation of Tonic and Phasic Motoneurons in Drosophila

“…Since phasic Is motoneurons have stronger synapses with higher P r active zones than their tonic Ib counterparts at the third instar stage ( Kurdyak et al, 1994 ; Lnenicka and Keshishian, 2000 ; Lu et al, 2016 ; Newman et al, 2017 ; Genç and Davis, 2019 ; Karunanithi et al, 2020 ), we examined Is synaptic maturation given their shorter developmental time window compared with the pre-existing Ib input. Presynaptic bouton and AZ number at motoneuron NMJs increase throughout larval development to help maintain normal levels of depolarization during the rapid expansion of muscle size during this period.…”

Tracking single molecule dynamics in the anesthetizedDrosophilabrain

“…A Sx1a gain-of-function mutation was found to confer resistance to volatile general anesthetics in the nematode Caenorhabditis elegans (van Swinderen et al, 1999) as well as Drosophila flies (Troup et al, 2019), suggesting a potential presynaptic target mechanism for these drugs. Consistent with this view, electrophysiological recordings from the fly neuromuscular junction reveal decreased quantal release under propofol anesthesia (Karunanithi et al, 2020), and super-resolution microscopy showed that propofol immobilizes Syx1a in nanoclusters that are potentially unavailable to form SNARES (Bademosi et al, 2018b). This mechanism has been hypothesized to explain the well-documented reduction of chemical neurotransmission observed in the presence of some general anesthetics (Bademosi et al, 2018b; Baumgart et al, 2015; Covarrubias et al, 2015; Hemmings et al, 2005; Herring et al, 2011, 2009; Karunanithi et al, 2020; Zalucki et al, 2015), but it remains unclear whether this effect is common to all classes of general anesthetics (i.e., volatile and intravenous), and if it is also evident in central synapses in the brain.…”

“…Consistent with this view, electrophysiological recordings from the fly neuromuscular junction reveal decreased quantal release under propofol anesthesia (Karunanithi et al, 2020), and super-resolution microscopy showed that propofol immobilizes Syx1a in nanoclusters that are potentially unavailable to form SNARES (Bademosi et al, 2018b). This mechanism has been hypothesized to explain the well-documented reduction of chemical neurotransmission observed in the presence of some general anesthetics (Bademosi et al, 2018b; Baumgart et al, 2015; Covarrubias et al, 2015; Hemmings et al, 2005; Herring et al, 2011, 2009; Karunanithi et al, 2020; Zalucki et al, 2015), but it remains unclear whether this effect is common to all classes of general anesthetics (i.e., volatile and intravenous), and if it is also evident in central synapses in the brain. In recent electrophysiological work, we have shown that clinically relevant concentrations of the intravenous agent propofol decreases the number active release sites at glutamatergic synapses in the fly larval neuromuscular junction, while a propofol analog has no such effect (Karunanithi et al, 2020).…”

“…Mechanisms of chemical neurotransmission have become increasingly understood over the past several decades (Baker and Hughson, 2016; Han et al, 2017; Südhof, 2012; Sudhof and Rothman, 2009) and this knowledge has uncovered novel hypotheses for how neurotransmission might be compromised by general anesthetics (Bademosi et al, 2018b; Baumgart et al, 2015; Hemmings et al, 2019, 2005; Humphrey et al, 2007; Karunanithi et al, 2020; Troup et al, 2019; van Swinderen and Kottler, 2014). A key recent advancement aiding our understanding of synaptic function is the development super resolution microscopy, which allows for the visualization of proteins and molecules below the diffraction limit of light (Betzig et al, 2006; Willig et al, 2006).…”

“…This mechanism has been hypothesized to explain the well-documented reduction of chemical neurotransmission observed in the presence of some general anesthetics (Bademosi et al, 2018b; Baumgart et al, 2015; Covarrubias et al, 2015; Hemmings et al, 2005; Herring et al, 2011, 2009; Karunanithi et al, 2020; Zalucki et al, 2015), but it remains unclear whether this effect is common to all classes of general anesthetics (i.e., volatile and intravenous), and if it is also evident in central synapses in the brain. In recent electrophysiological work, we have shown that clinically relevant concentrations of the intravenous agent propofol decreases the number active release sites at glutamatergic synapses in the fly larval neuromuscular junction, while a propofol analog has no such effect (Karunanithi et al, 2020). This suggests a failure in the recruitment of synaptic release machinery components under propofol, which would be consistent with our finding that propofol immobilizes syntaxin1A in fly larval synapses (Bademosi et al, 2018b).…”