Proportional Downscaling of Glutamatergic Release Sites by the General Anesthetic Propofol at Drosophila Motor Nerve Terminals.

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 µm) 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.

Syntaxin1A Neomorphic Mutations Promote Rapid Recovery from Isoflurane Anesthesia in Drosophila melanogaster.

BACKGROUND: Mutations in the presynaptic protein syntaxin1A modulate general anesthetic effects in vitro and in vivo. Coexpression of a truncated syntaxin1A protein confers resistance to volatile and intravenous anesthetics, suggesting a target mechanism distinct from postsynaptic inhibitory receptor processes. Hypothesizing that recovery from anesthesia may involve a presynaptic component, the authors tested whether syntaxin1A mutations facilitated recovery from isoflurane anesthesia in Drosophila melanogaster. METHODS: A truncated syntaxin1A construct was expressed in Drosophila neurons. The authors compared effects on isoflurane induction versus recovery in syntaxin1A mutant animals by probing behavioral responses to mechanical stimuli. The authors also measured synaptic responses from the larval neuromuscular junction using sharp intracellular recordings, and performed Western blots to determine whether the truncated syntaxin1A is associated with presynaptic core complexes. RESULTS: Drosophila expressing a truncated syntaxin1A (syx, n = 40) were resistant to isoflurane induction for a behavioral responsiveness endpoint (ED50 0.30 ± 0.01% isoflurane, P < 0.001) compared with control (0.240 ± 0.002% isoflurane, n = 40). Recovery from isoflurane anesthesia was also faster, with syx-expressing flies showing greater levels of responsiveness earlier in recovery (reaction proportion 0.66 ± 0.48, P < 0.001, n = 68) than controls (0.22 ± 0.42, n = 68 and 0.33 ± 0.48, n = 66). Measuring excitatory junction potentials of larvae coexpressing the truncated syntaxin1A protein showed a greater recovery of synaptic function, compared with controls (17.39 ± 3.19 mV and 10.29 ± 4.88 mV, P = 0.014, n = 8 for both). The resistance-promoting truncated syntaxin1A was not associated with presynaptic core complexes, in the presence or absence of isoflurane anesthesia. CONCLUSIONS: The same neomorphic syntaxin1A mutation that confers isoflurane resistance in cell culture and nematodes also produces isoflurane resistance in Drosophila. Resistance in Drosophila is, however, most evident at the level of recovery from anesthesia, suggesting that the syntaxin1A target affects anesthesia maintenance and recovery processes rather than induction. The absence of truncated syntaxin1A from the presynaptic complex suggests that the resistance-promoting effect of this molecule occurs before core complex formation.

Trapping of Syntaxin1a in Presynaptic Nanoclusters by a Clinically Relevant General Anesthetic.

Propofol is the most commonly used general anesthetic in humans. Our understanding of its mechanism of action has focused on its capacity to potentiate inhibitory systems in the brain. However, it is unknown whether other neural mechanisms are involved in general anesthesia. Here, we demonstrate that the synaptic release machinery is also a target. Using single-particle tracking photoactivation localization microscopy, we show that clinically relevant concentrations of propofol and etomidate restrict syntaxin1A mobility on the plasma membrane, whereas non-anesthetic analogs produce the opposite effect and increase syntaxin1A mobility. Removing the interaction with the t-SNARE partner SNAP-25 abolishes propofol-induced syntaxin1A confinement, indicating that syntaxin1A and SNAP-25 together form an emergent drug target. Impaired syntaxin1A mobility and exocytosis under propofol are both rescued by co-expressing a truncated syntaxin1A construct that interacts with SNAP-25. Our results suggest that propofol interferes with a step in SNARE complex formation, resulting in non-functional syntaxin1A nanoclusters.

Local Versus Global Effects of Isoflurane Anesthesia on Visual Processing in the Fly Brain.

What characteristics of neural activity distinguish the awake and anesthetized brain? Drugs such as isoflurane abolish behavioral responsiveness in all animals, implying evolutionarily conserved mechanisms. However, it is unclear whether this conservation is reflected at the level of neural activity. Studies in humans have shown that anesthesia is characterized by spatially distinct spectral and coherence signatures that have also been implicated in the global impairment of cortical communication. We questioned whether anesthesia has similar effects on global and local neural processing in one of the smallest brains, that of the fruit fly (Drosophila melanogaster). Using a recently developed multielectrode technique, we recorded local field potentials from different areas of the fly brain simultaneously, while manipulating the concentration of isoflurane. Flickering visual stimuli ('frequency tags') allowed us to track evoked responses in the frequency domain and measure the effects of isoflurane throughout the brain. We found that isoflurane reduced power and coherence at the tagging frequency (13 or 17 Hz) in central brain regions. Unexpectedly, isoflurane increased power and coherence at twice the tag frequency (26 or 34 Hz) in the optic lobes of the fly, but only for specific stimulus configurations. By modeling the periodic responses, we show that the increase in power in peripheral areas can be attributed to local neuroanatomy. We further show that the effects on coherence can be explained by impacted signal-to-noise ratios. Together, our results show that general anesthesia has distinct local and global effects on neuronal processing in the fruit fly brain.

Syntaxin1A-mediated Resistance and Hypersensitivity to Isoflurane in Drosophila melanogaster.

BACKGROUND: Recent evidence suggests that general anesthetics activate endogenous sleep pathways, yet this mechanism cannot explain the entirety of general anesthesia. General anesthetics could disrupt synaptic release processes, as previous work in Caenorhabditis elegans and in vitro cell preparations suggested a role for the soluble NSF attachment protein receptor protein, syntaxin1A, in mediating resistance to several general anesthetics. The authors questioned whether the syntaxin1A-mediated effects found in these reductionist systems reflected a common anesthetic mechanism distinct from sleep-related processes. METHODS: Using the fruit fly model, Drosophila melanogaster, the authors investigated the relevance of syntaxin1A manipulations to general anesthesia. The authors used different behavioral and electrophysiological endpoints to test the effect of syntaxin1A mutations on sensitivity to isoflurane. RESULTS: The authors found two syntaxin1A mutations that confer opposite general anesthesia phenotypes: syxH3-C, a 14-amino acid deletion mutant, is resistant to isoflurane (n = 40 flies), and syxKARRAA, a strain with two amino acid substitutions, is hypersensitive to the drug (n = 40 flies). Crucially, these opposing effects are maintained across different behavioral endpoints and life stages. The authors determined the isoflurane sensitivity of syxH3-C at the larval neuromuscular junction to assess effects on synaptic release. The authors find that although isoflurane slightly attenuates synaptic release in wild-type animals (n = 8), syxH3-C preserves synaptic release in the presence of isoflurane (n = 8). CONCLUSION: The study results are evidence that volatile general anesthetics target synaptic release mechanisms; in addition to first activating sleep pathways, a major consequence of these drugs may be to decrease the efficacy of neurotransmission.