Tracking single molecule dynamics in the anesthetized Drosophila brain

Super-resolution microscopy provides valuable insight for understanding the nanoscale organization within living tissue, although this method is typically restricted to cultured or dissociated cells. Here, we develop a method to track the mobility of individual proteins in ex vivo adult Drosophila melanogaster brains, focusing on a key component of the presynaptic release machinery, syntaxin1A. We show that individual syntaxin1A dynamics can be reliably tracked within neurons in the whole fly brain, and that the mobility of syntaxin1A molecules increases following conditional neural stimulation. We then apply this preparation to the problem of general anesthesia, to address how different anesthetics might affect single molecule dynamics in intact brain synapses. We find that propofol, etomidate, and isoflurane significantly impair syntaxin1A mobility, while ketamine and sevoflurane have little effect. Resolving single molecule dynamics in intact fly brains provides a novel approach to link localized molecular effects with systems-level phenomena such as general anesthesia. Impact statement A new approach to track the mobility of individual molecules using intact fly brains reveals a common presynaptic effect for different intravenous and volatile general anesthetics.


Introduction 1 2
Mechanisms of chemical neurotransmission have become increasingly understood over 3 the past several decades (Baker and Hughson, 2016;Han et al., 2017;Südhof, 2012;4 Sudhof and Rothman, 2009) and this knowledge has uncovered novel hypotheses for how 5 neurotransmission might be compromised by general anesthetics (Bademosi et al., 2018b;6 Baumgart et al., 2015;Hemmings et al., 2019Hemmings et al., , 2005Humphrey et al., 2007;Karunanithi 7 et al., 2020;Troup et al., 2019;van Swinderen and Kottler, 2014). A key recent 8 advancement aiding our understanding of synaptic function is the development super 9 resolution microscopy, which allows for the visualization of proteins and molecules 10 below the diffraction limit of light (Betzig et al., 2006;Willig et al., 2006). Super-11 resolution microscopy has provided novel insight on the nanoscale structure and 12 dynamics of key components of the presynaptic release machinery, such as syntaxin1A 13 (Bademosi et al., 2016;Reddy-Alla et al., 2017;Ullrich et al., 2015). Photoactivatable Eos-tagged proteins, dual color illumination in a total internal reflection (TIRF) (Axelrod, 22 2001) or highly inclined and laminated optical (HILO) (Tokunaga et al., 2008) sheet 23 configuration is employed to simultaneously record and stochastically photoconvert Eos 24 fluorophores in cultured cells or dissociated neurons (Manzo and Garcia-Parajo, 2015). 25 However, there is comparatively little information on single molecule dynamics in more 26 complex living tissue, such as animal brains.

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Recently, we have described single molecule imaging in motor nerve terminals of filleted 29 larvae of the fruit fly Drosophila melanogaster (Bademosi et al., 2018a(Bademosi et al., , 2016. In that 30 study we tagged the pre-synaptic protein syntaxin1A (Sx1a) with photoconvertible 31 mEos2 and found that genetic activation of motoneurons resulted in increased mobility of 32 Sx1A in the motor nerve terminals, suggesting increased mobilization of the presynaptic 33 machinery when neurons are activated. Sx1a is necessary for the docking and fusion of 34 neurotransmitter-containing vesicles, and is a component of the SNARE complex along 35 with its binding partners SNAP25 and VAMP2 (Südhof, 2012). Sx1a function is highly 36 conserved in all animals (Bennett et al., 1992;Ferro-Novick and Jahn, 1994;Sudhof and 37 Rizo, 2011), with mutations in the protein often implicated in synaptic communication 38 defects and lethality (Fergestad et al., 2001;Fujiwara et al., 2006;Kofuji et al., 2017;39 Saifee et al., 1998;Schulze et al., 1995;Vardar et al., 2016). A Sx1a gain-of-function 40 mutation was found to confer resistance to volatile general anesthetics in the nematode 41 Caenorhabditis elegans (van Swinderen et al., 1999) as well as Drosophila flies (Troup et   42 al., 2019), suggesting a potential presynaptic target mechanism for these drugs. nanoclusters that are potentially unavailable to form SNARES (Bademosi et al., 2018b). 2 This mechanism has been hypothesized to explain the well-documented reduction of 3 chemical neurotransmission observed in the presence of some general anesthetics 4 (Bademosi et al., 2018b;Baumgart et al., 2015;Covarrubias et al., 2015;Hemmings et 5 al., 2005;Herring et al., 2011Herring et al., , 2009Karunanithi et al., 2020;Zalucki et al., 2015), but it 6 remains unclear whether this effect is common to all classes of general anesthetics (i.e., 7 volatile and intravenous), and if it is also evident in central synapses in the brain. In 8 recent electrophysiological work, we have shown that clinically relevant concentrations 9 of the intravenous agent propofol decreases the number active release sites at 10 glutamatergic synapses in the fly larval neuromuscular junction, while a propofol analog 11 has no such effect (Karunanithi et al., 2020). This suggests a failure in the recruitment of 12 synaptic release machinery components under propofol, which would be consistent with 13 our finding that propofol immobilizes syntaxin1A in fly larval synapses (Bademosi et al., 14 2018b). What remains unknown is if a similar effect is evident in central synapses, which 15 would be the more relevant targets for inducing and/or maintaining general anesthesia.

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Here, we adapt super resolution imaging techniques to the extracted adult fly brain and 18 utilize this novel approach to test whether diverse intravenous and volatile general 19 anesthetics might share a common presynaptic mechanism in the central nervous system. 20 We found that, similar to Drosophila larval neuromuscular junction (Bademosi et al.,21 2016), the mobility of Sx1a molecules in the adult brain is increased upon neuronal 22 stimulation, thereby providing a physiologically relevant setting to probe for general 23 anesthetic effects in intact brain tissue. We exposed fly brains to diverse volatile and 24 intravenous general anesthetics, to determine if syntaxin1A mobility is also affected in 25 intact brain tissue by some of these commonly used drugs.

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Imaging syntaxin1A mobility in the adult fly brain 31 32 We employed single particle tracking PALM (sptPALM) to image and track individual   Figure 1D). When observing the brain at 100x 44 magnification, the point spread function (PSF) overlap of the unconverted green form of 45 mEos2 does not allow for the resolution of individual molecules or structures within the 1 fly brain ( Figure 1E). Upon exposure to a low intensity ultraviolet (UV, 405 nm) 2 photoconverting stimulus, stochastically switched red mEos2 molecules can be visualized 3 sparsely ( Figure 1F). In order to confirm that we were imaging mEos2 molecules, we 4 compared spot counts in brains that had no UV exposure and saw a significant increase in 5 single molecule detection with photoconversion ( Figure 1 -figure supplement 3). At 32 6 msec exposure time, Sx1a-mEos2 molecules can be seen moving inside of neurons of the 7 fly brain (Video supplement 1). Neural structures in the fly brain become evident after 8 performing a maximum projection of a time series of PALM experiments ( Figure 1G), 9 confirming that Sx1a-mEos2 is confined.

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In order to characterize the mobility of individual tagged proteins, we performed single  23 24 To validate the reproducibility of our approach, we compared Sx1a-mEos2 mobility 25 across successive recording sessions from the same brains. We recorded from different 26 brain regions (Figure 2A-C) and from the same brain region ( Figure 2F-H). We observed 27 considerable variability in Sx1a-mEos2 mobility across experiments and brain regions 28 ( Figure 2D,E), consistent with the large range in MSDs observed in our first dataset 29 ( Figure 1I). Crucially, successive recordings from the same region (top right of the 30 central brain, approximately in the lateral protocerebrum) revealed a high level of 31 consistency in the number of localizations, trajectories, and MSD values within a 32 recording site ( Figure 2I). This shows that results are repeatable in the same location, but 33 also that some variability in diffusion coefficients exists across experiments in different 34 brains ( Figure 2J). Importantly, successive recordings from the same brain region  supplement 2). In addition to this, imaging only HL3.1 solution without any brain tissue 7 revealed highly mobile bright spots that could be localized, but not tracked utilizing our 8 SPA software (Video supplement 3). We next investigated if we could increase Sx1a-9 mEos2 mobility when we stimulated neurons. In previous work, we have shown that supplement 1). When neurons of the fly brain were stimulated, we observed a consistent 20 and significant increase in Sx1a-mEos2 mobility compared to baseline unstimulated 21 conditions (n = 13, p = 0.0002, Wilcoxon test, Figure 3C,D). In contrast, no significant 22 increase in Sx1a-mEos2 mobility was observed at the elevated temperature in control 23 brains that did not express dTrpA1 ( Figure 3E,F).

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General anesthetics restrict syntaxin1A mobility in brain neurons 26 27 Having conditionally increased Sx1a-mEos2 mobility in the fly brain, we next sought to 28 pharmacologically perturb this effect in the same preparation. We have previously shown 29 that the intravenous general anesthetics propofol and etomidate decrease Sx1a-mEos2 Sx1a is highly dynamic in the adult fly brain, with increased mobility following neural 3 stimulation and decreased mobility under general anesthetic exposure. This confirms and 4 expands findings in other model systems (Bademosi et al., 2018b(Bademosi et al., , 2016, and shows that 5 some commonly used intravenous and volatile general anesthetics might share a 6 presynaptic target mechanism centered on Sx1A.

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In conclusion, we find that different classes of general anesthetics, such as propofol and 9 isoflurane, have similar effects on the mobility of a presynaptic protein, whereas others 10 such as ketamine and sevoflurane have little effect. General anesthesia has been largely 11 explained as a post-synaptic phenomenon linked to potentiation of inhibitory ion channels 12 (Franks, 2008;Franks and Lieb, 1994;Masiulis et al., 2019). Our results suggest a 13 presynaptic mechanism for some of these drugs, which is consistent with other work done  We thank Adekunle Bademosi and Merja Joensuu for critical discussions about the work, 10 Rumelo Amor for help with microscopy, and the van Swinderen lab for feedback on the 11 project. This study was funded NHMRC GNT1065715 and GNT1164879 (to BvS) and 12 the Zeiss ELYRA microscope was funded by ARC LIEF LE130100078.

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For fixed brain imaging, brains were dissected as usual and then fixed in 4% Super resolution and photoactivatable localization microscopy 16 All imaging was performed on a standard Zeiss ELYRA PS.1 microscope fitted with a 17 Zeiss Plan-APOCHROMAT 100x 1.4nA oil immersion objective, a Zeiss FC12 definite 18 focus, and an iXon EMCCD 512x512 pixel camera (Andor, Oxford Instruments).

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Mounted brains were inverted so that the oil-objective touches the coverslip and the ROI 20 was navigated visually using bright-field illumination. Brains were imaged at a highly  0.05 was used. MSD presented is ± standard deviation (SD) and AUC data is ± 5-95th 36 percentile. 95% Confidence intervals (CI) were calculated around the mean.

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Data and Code Availability

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The datasets and code supporting the current study will be made available on a public 40 database upon publication.    Schematic of tracking particles in a 2D-sample over time, with links between frames 6 determined based on the relative distance (δ, distance) of a single particle to every other 7 particles (D) from one frame to the next. (E) Particle tracking is solved using a linear 8 assignment problem cost matrix, where the cost is the relative distance of a particle in frame 9 n to every other particle in frame n+1. A particle in frame n can have one of four outcomes 10 based on the localisation in the proceeding frame. A particle has a potential link (λ) to 11 another particle based on a maximum linking distance which if a particle in the proceeding the molecule, and if it is membrane bound or cytoplasmic. A particle can also either be the 17 29 start or the end of a trajectory, and a higher cost value is employed to determine if a particle 1 should be linked to another particle or not (α & β). (F) Example of the cost matrix utilised 2 to solve single particle tracking. The matrix is solved for least cost to link particles and 3 determine if a trajectory is at its beginning or its end (Adapted from Jaqaman et al. 2008 4 (Jaqaman et al., 2008)).

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The relatively high number of spots in the 561 nm condition is most likely due to the high 11 amount of auto-photoconversion after brain dissection under bright-field lights. Wilcoxon test, data is ± 5-95 th percentile).