Proportional Downscaling of Glutamatergic Release Sites by the General Anesthetic Propofol at 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 motor nerve terminals to characterize this effect.

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.

Using Drosophila to Understand General Anesthesia: From Synapses to Behavior.

Investigating mechanisms of general anesthesia requires access to multiple levels of neuronal function, from effects at individual synapses to responses in behaving animals. Drosophila melanogaster provides an excellent model to test different theories for general anesthesia because it offers robust methods for testing local as well as global target processes, in an animal that is similarly impacted by these diverse drugs as humans. Here, we outline methods to quantify two such endpoints, neurotransmission and behavioral responsiveness, focusing on the intravenous drug propofol.

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.

Flux of signalling endosomes undergoing axonal retrograde transport is encoded by presynaptic activity and TrkB.

Axonal retrograde transport of signalling endosomes from the nerve terminal to the soma underpins survival. As each signalling endosome carries a quantal amount of activated receptors, we hypothesized that it is the frequency of endosomes reaching the soma that determines the scale of the trophic signal. Here we show that upregulating synaptic activity markedly increased the flux of plasma membrane-derived retrograde endosomes (labelled using cholera toxin subunit-B: CTB) in hippocampal neurons cultured in microfluidic devices, and live Drosophila larval motor neurons.

Behavioral and electrophysiological analysis of general anesthesia in 3 background strains of Drosophila melanogaster.

General anesthetics achieve behavioral unresponsiveness via a mechanism that is incompletely understood. The study of genetic model systems such as the fruit fly Drosophila melanogaster is crucial to advancing our understanding of how anesthetic drugs render animals unresponsive. Previous studies have shown that wild-type control strains differ significantly in their sensitivity to general anesthetics, which potentially introduces confounding factors for comparing genetic mutations placed on these wild-type backgrounds.

Heat shock response and homeostatic plasticity.

Heat shock response and homeostatic plasticity are mechanisms that afford functional stability to cells in the face of stress. Each mechanism has been investigated independently, but the link between the two has not been extensively explored. We explore this link.

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.

Fos and Jun potentiate individual release sites and mobilize the reserve synaptic vesicle pool at the Drosophila larval motor synapse.

In all nervous systems, short-term enhancement of transmitter release is achieved by increasing the weights of unitary synapses; in contrast, long-term enhancement, which requires nuclear gene expression, is generally thought to be mediated by the addition of new synaptic vesicle release sites. In Drosophila motor neurons, induction of AP-1, a heterodimer of Fos and Jun, induces cAMP- and CREB-dependent forms of presynaptic enhancement. Light and electron microscopic studies indicate that this synaptic enhancement is caused by increasing the weight of unitary synapses and not through the insertion of additional release sites.

Morphological and functional effects of altered cysteine string protein at the Drosophila larval neuromuscular junction.

The synaptic vesicle-associated cysteine string protein (CSP) is critical for neurotransmitter release at the neuromuscular junction (NMJ) of Drosophila, where the approximately 4% of mutant flies lacking CSP that survive to adulthood exhibit spastic jumping and shaking, temperature-sensitive paralysis, and premature death. Previously, it has been shown that CSP is also required for nerve terminal growth and the prevention of neurodegeneration in Drosophila and mice. At larval csp null mutant NMJs of Drosophila, intracellular recordings from the muscle showed that evoked release is significantly reduced at room temperature.

Thermoprotection of synaptic transmission in a Drosophila heat shock factor mutant is accompanied by increased expression of Hsp83 and DnaJ-1.

In Drosophila larvae, acquired synaptic thermotolerance after heat shock has previously been shown to correlate with the induction of heat shock proteins (Hsps) including HSP70. We tested the hypothesis that synaptic thermotolerance would be significantly diminished in a temperature-sensitive strain (Drosophila heat shock factor mutant hsf4), which has been reported not to be able to produce inducible Hsps in response to heat shock. Contrary to our hypothesis, considerable thermoprotection was still observed at hsf4 larval synapses after heat shock.

Quantal size and variation determined by vesicle size in normal and mutant Drosophila glutamatergic synapses.

Quantal size and variation at chemical synapses could be determined presynaptically by the amount of neurotransmitter released from synaptic vesicles or postsynaptically by the number of receptors available for activation. We investigated these possibilities at Drosophila glutamatergic neuromuscular synapses formed by two separate motor neurons innervating the same muscle cell. At wild-type synapses of the two neurons we found a difference in quantal size corresponding to a difference in mean synaptic vesicle volume.

Diversification of synaptic strength: presynaptic elements.

Synapses are not static; their performance is modified adaptively in response to activity. Presynaptic mechanisms that affect the probability of transmitter release or the amount of transmitter that is released are important in synaptic diversification. Here, we address the diversity of presynaptic performance and its underlying mechanisms: how much of the variation can be accounted for by variation in synaptic morphology and how much by molecular differences? Significant progress has been made in defining presynaptic structural contributions to synaptic strength; by contrast, we know little about how presynaptic proteins produce normally observed functional differentiation, despite abundant information on presynaptic proteins and on the effects of their individual manipulation.

Enhancement of presynaptic performance in transgenic Drosophila overexpressing heat shock protein Hsp70.

Prior heat shock confers protection to Drosophila synapses during subsequent heat stress by stabilizing quantal size and reducing the decline of quantal emission at individual synaptic boutons. The major heat shock protein Hsp70, which is strongly induced by high temperatures in Drosophila, may be responsible for this synaptic protection. To test this hypothesis, we investigated synaptic protection and stabilization at larval neuromuscular junctions of transgenic Drosophila which produce more than the normal amount of Hsp70 in response to heat shock.