Axonal differentiation of retinal bipolar cells has largely been
studied by comparing the morphology of these interneurons in fixed tissue
at different ages. To better understand how bipolar axonal terminals
develop in vivo, we imaged fluorescently labeled cells in the
zebrafish retina using time-lapse confocal and two photon microscopy.
Using the upstream regulatory sequences from the nyx gene that
encodes nyctalopin, we constructed a transgenic fish in which a subset of
retinal bipolar cells express membrane targeted yellow fluorescent protein
(MYFP). Axonal terminals of these YFP-labeled bipolar cells laminated
primarily in the inner half of the inner plexiform layer, suggesting that
they are likely to be ON-bipolar cells. Transient expression of MYFP in
isolated bipolar cells indicates that two or more subsets of bipolar
cells, with one or two terminal boutons, are labeled. Live imaging of
YFP-expressing bipolar cells in the nyx::MYFP transgenic fish at
different ages showed that initially, filopodial-like structures extend
and retract from their primary axonal process throughout the inner
plexiform layer (IPL). Over time, filopodial exploration becomes
concentrated at discrete foci prior to the establishment of large terminal
boutons, characteristic of the mature form. This sequence of axonal
differentiation suggests that synaptic targeting by bipolar cell axons may
involve an early process of trial and error, rather than a process of
directed outgrowth and contact. Our observations represent the first
in vivo visualization of axonal development of bipolar cells in a
vertebrate retina.
