Abstract
Purpose::
The current concept of synapse assembly at conventional chemical synapses postulates "active zone precursor vesicles" that deliver active zone protein complexes to nascent synapses, leading to the rapid formation of the presynapse (Zhai et al. 2001, Neuron 29:131-143). A specialization of the cytomatrix at the active zone is the plate-like ribbon in photoreceptor cells of the retina. In our group we study the developmental assembly of this complex synapse.
Methods::
With immunocytochemistry and confocal laser-scanning microscopy we have examined the molecular assembly of the presynaptic ribbon complex, and using electron microscopy we have studied the ultrastructural appearance of the ribbon during photoreceptor synaptogenesis.
Results::
In the mouse retina, immunofluorescent aggregates of the presynaptic proteins Bassoon, Piccolo, RIBEYE and RIM1 are present in the neuroblast layer very early in postnatal development. These proteins are transported in a complex along the growing photoreceptor axon to the future ribbon synaptic site. Proteins of the arciform density compartment are not part of this complex. Ultrastructurally, the aggregates appear as round, non-membranous, electron-dense matrix, surrounded by tethered synaptic vesicle-like structures. 3D-reconstruction shows that most of these profiles are spheres with up to 200 nm in diameter. With increasing age a continuous decrease in the number of precursor profiles is accompanied by an increase in the number of ribbons. The 80 nm "active zone precursor vesicles", as described for conventional synapses, could not be identified so far in photoreceptor synaptogenesis.
Conclusions::
From our data we conclude that the mature photoreceptor ribbon is formed from non-membranous "precursor spheres" which are transported separately from the proteins of the arciform density compartment to the future ribbon synaptic site. To investigate the specific contribution of the various presynaptic proteins to ribbon formation, we will knockdown every single one during synaptogenesis by RNA interference (RNAi). RNAi knockdown experiments and subsequent immunocytochemical and biochemical analysis of the photoreceptors will enable us to unravel the developmental assembly of the photoreceptor ribbon synapse.
Keywords: retina • synapse • development