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

Abstract

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. However, it remained unclear whether the reduction in evoked release might be due to a loss of synaptic boutons, loss of synapses, and alterations in trafficking of vesicles to synapses. To resolve these issues, we have examined synaptic structure and function of csp null mutant NMJs at the level of single boutons. csp null mutations proportionally reduce the number of synaptic boutons of both motor neurons (1s and 1b) innervating larval muscles 6 and 7, while the number of synapses per bouton remains normal. However, focal recordings from individual synaptic boutons show that nerve-evoked neurotransmitter release is also impaired in both 1s and 1b boutons. Further, our ultrastructural analyses show that the reduction in evoked release at low stimulation frequencies is not due to a loss of synapses or to alterations in docked vesicles at synapses. Together, these data suggest that CSP promotes synaptic growth and evoked neurotransmitter release by mechanistically independent signaling pathways.

Mutations in palmitoyl-protein thioesterase 1 alter exocytosis and endocytosis at synapses in Drosophila larvae

“…Several genes known to play a role in this process also play a role in the structural development of the synapse. 33 To determine if there were structural abnormalities associated with these mutants, we examined synaptic development in Ppt1 third instar larvae. Staining of the Ppt1 deficiency mutant larval neuromuscular junctions showed no gross morphological changes, and bouton number (100 +/-7.02), as shown by antisynaptotagmin staining, was not significantly altered as compared with wild-type controls (116 +/-4.60; p = 0.11, Nonparametric Mann Whitney U Test) ( Fig.…”

Cysteine-String Protein’s Role at Synapses

“…8.4). By P30, the reduction of evoked release is quantitatively similar to that seen at the CSPa mutant Calyx of Held (see below), dCSP mutant NMJs, and frog NMJs upon injection of CSP antibodies Zinsmaier et al 1994;Poage et al 1999;Dawson-Scully et al 2000;Fernandez-Chacon et al 2004;Dawson-Scully et al 2007).…”

“…With increasing temperature, tetanically evoked presynaptic Ca 2+ signals are increasingly larger at dCSP mutant terminals than in control (Dawson-Scully et al 2000). The elevated Ca 2+ levels are likely due to a defect in presynaptic Ca 2+ clearance, since tetanicallyinduced Ca 2+ signals exhibit a prolonged decay at higher temperatures, and because single action-potential induced Ca 2+ signals are normal at any examined temperature (Bronk et al 2005;Dawson-Scully et al 2007). Consistently, presynaptic Ca 2+ levels at rest are normal at 24°C but elevated at 34°C (Dawson-Scully et al 2000).…”

“…High-frequency stimulation partially rescues 10 Hz the temperature-sensitive block of release observed at 0.2 Hz stimulation while increasing the extracellular Ca 2+ to 4 mM fully restores faithful and stable synaptic transmission at 0.2 Hz stimulation. Reprinted from Umbach et al (1994) and Dawson-Scully et al (2000) with permission from Elsevier and the Society for Neuroscience, respectively by ~70% (Dawson-Scully et al 2007). Much in contrast to photoreceptor terminals of adult dCSP mutant animals, larval NMJs show no signs of neurodegeneration and no loss of synaptic vesicles Dawson-Scully et al 2007).…”

“…Major insights into the neuroprotective role of CSP came from genetic studies in flies and mice Zinsmaier et al 1994;Heckmann et al 1997;Umbach and Gundersen 1997;Eberle et al 1998;Ranjan et al 1998;Umbach et al 1998;Dawson-Scully et al 2000;Bronk et al 2001;Fernandez-Chacon et al 2004;Bronk et al 2005;Chandra et al 2005;Schmitz et al 2006;Dawson-Scully et al 2007). In Drosophila, deletion of the entire dCSP gene causes temperature-sensitive paralysis and semi-lethality.…”