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Deletion/blockade of Septin 5 converts “microdomain” to “nanodomain” coupling between Ca2+ channels and synaptic vesicles at a developing central synapse

Yi-Mei Yang1, 2*, M. J. Fedchyshyn1, 2, G. Grande1, 2, J. Aitoubah1, 2, C. W. Tsang3, 4, H. Xie3, 4, W. S. Trimble3, 4 and Lu-Yang Wang1, 2
  • 1 Hospital for Sick Children, Program in Neurosciences and Mental Health, Canada
  • 2 University of Toronto, Department of Physiology, Canada
  • 3 University of Toronto, Department of Biochemistry, Canada
  • 4 Hospital for Sick Children, Cell Biology, Canada

Neurotransmitter release critically depends on spatial coupling modalities between voltage-gated Ca2+ channels (VGCCs) and synaptic vesicles (SVs) near active zones (AZs) of nerve terminals, however molecular substrates regulating their physical proximity are unknown. Ultrastructural analyses reveal that a filamentous mesh of projections emanating from the AZ appears to encircle and link SVs closely to the presynaptic membrane. Filamentous protein Septin 5 (Sept5) or Sept5 containing heteroligomers have been shown to inhibit SNARE-mediated vesicular release in secretary cells, leading to the hypothesis Sept5 may act as a molecular “brake” preventing SV association with the t-SNARE complex and interfering SV release. By performing immuno-fluorescence and electron microscopy (EM), and electrophysiological recordings from wild-type (Sept5+/+) and Sept5 knockout (Sept5-/-) synapses, we investigated the potential roles of Sept5 in regulating subsynaptic organization and function at the mouse calyx of Held synapse. At this synapse, loose “microdomain” couplings between VGCCs and SVs predominate at the early postnatal stage but developmentally evolve to tight “nanodomain” couplings to boost quantal output. Antibody co-localization analyses showed that Sept5 is abundantly expressed in immature Sept5+/+ but not in Sept5-/- calyces, with a preferential localization to the release side facing synaptic cleft while SV marker VGlut2 is homogeneously distributed. In line with this, EM analyses revealed more docked vesicles on AZs in Sept5-/- calyces than in Sept5+/+ calyces of immature synapses, suggesting a deletion of Sept5 allows more SVs to tightly associate with AZs where VGCCs presumably reside. Simultaneous paired pre- and post-synaptic recordings demonstrated that immature Sept5-/- synapses display much less sensitivity to slow Ca2+ buffer EGTA, and require fewer VGCCs (or lower Ca2+ channel/domain cooperativity, m) to trigger single fusion events than Sept5+/+ synapses. These observations indicate that elimination of Sept5 gene imparts immature synapses striking morphological and functional features of “nanodomain” couplings, reminiscent of those in mature synapses. Furthermore, functional block of Sept5 with peptide (Borg3) or antibody (SP20) injections into immature Sept5+/+ nerve terminals can acutely transform microdomain to nanodomain couplings, as evidenced by a significant increase in quantal output and reciprocally a decrease in m. Our results collectively suggest that Sept5 is one of the key molecular substrates that differentiate distinct release modalities at this and perhaps other central synapses.


Supported by CIHR, OGS and SickKids RESTRACOMP

Conference: B.R.A.I.N. platform in Physiology poster day 2009, Toronto, ON, Canada, 16 Dec - 16 Dec, 2009.

Presentation Type: Poster Presentation

Topic: Poster presentations

Citation: Yang Y, Fedchyshyn MJ, Grande G, Aitoubah J, Tsang CW, Xie H, Trimble WS and Wang L (2009). Deletion/blockade of Septin 5 converts “microdomain” to “nanodomain” coupling between Ca2+ channels and synaptic vesicles at a developing central synapse. Front. Neurosci. Conference Abstract: B.R.A.I.N. platform in Physiology poster day 2009. doi: 10.3389/conf.neuro.03.2009.17.054

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Received: 18 Dec 2009; Published Online: 18 Dec 2009.

* Correspondence: Yi-Mei Yang, Hospital for Sick Children, Program in Neurosciences and Mental Health, Toronto, Canada, yimeiyang@hotmail.com