Acute genetic perturbation of exocyst function in the rat calyx of Held impedes structural maturation, but spares synaptic transmission

Abstract

The exocyst is an octameric protein complex mediating polarized secretion by tethering vesicles to target membranes. In non-vertebrate neurons, the exocyst has been associated with constitutive membrane addition at growth cones and nerve terminals, but its function in synaptic vesicle trafficking at mammalian nerve terminals remains unclear. Here, we examined the role of the exocyst complex in immature postnatal day (P)13 and mature P21 rat calyces of Held. Exo70, an exocyst subunit conferring membrane anchoring of the complex, was tagged with green fluorescent protein (GFP) and overexpressed as a full-length subunit or as a dominant-negative C-terminally truncated variant (Exo70ΔC) disrupting membrane targeting. In vivo expression of the Exo70 subunits in the calyx was achieved by stereotaxic adeno-associated virus-mediated gene transfer into globular bushy cells of the rat ventral cochlear nucleus at P2. Overexpression of dominant-negative Exo70ΔC, but not full-length Exo70, decreased the structural complexity and volume of calyces, as assayed by confocal microscopy and three-dimensional reconstructions. The distribution of active zones and synaptic vesicles remained unaffected. Neither perturbation changed the characteristics of spontaneous and evoked neurotransmitter release, short-term depression or recovery from depression. Together, these data suggest that in central mammalian synapses, the exocyst complex mediates the addition of membrane during postnatal presynaptic maturation, but does not function as a tethering complex in local recycling of vesicles within the synaptic vesicle cycle.