Fig. 5. Trans-synaptic labeling of Mauthner neurons reveals connectivity defects with space cadet presynaptic spiral fiber neurons. (A) The calcium indicator calcium green dextran was injected into the ventral spinal cord to label Mauthner neurons. (B) In larvae with retrogradely labeled Mauthner neurons, the right Mauthner cell was injected with neurobiotin. In most larvae, the injected Mauthner cell soma died within 2-18 hours of neurobiotin injection, presumably owing to phototoxicity effects when CGD loaded neurons were exposed to intense light during neurobiotin injection. Therefore, the injected Mauthner cell soma is not visible. Deliberate injection of neurobiotin adjacent to the Mauthner cell did not label any neurons away from the injection site. (C-E) Wild-type larvae labeled with neurobiotin (C,E) or neurobiotin and 3A10 (D). (C) Injection into one wild-type Mauthner cell (M, outlined) labeled distinct sets of ipsilateral and contralateral neurons, most notably commissural spiral fiber neurons (arrows). (D) 3A10 antibody staining confirming that the two rhombomere 3 commissures contain spiral fiber axons. (E) High magnification view focused on spiral fiber cell bodies and the two commissures. (F-H) In space cadet larvae, neurobiotin injection (F,H at high magnification) revealed a similar distribution of labeled neurons located at the same level or caudally to the Mauthner cells. In contrast, neurobiotin labeled spiral fiber neurons and axons, as well as 3A10-labeled commissures are absent, demonstrating that in space cadet larvae spiral fibers are not connected to their synaptic target, the Mauthner cell.