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The lipid phosphatase synaptojanin 1 (synj1) is required for the disassembly of clathrin coats on endocytic compartments. In neurons such activity is necessary for the recycling of endocytosed membrane into synaptic vesicles. Mutations in zebrafish synj1 have been shown to disrupt the activity of ribbon synapses in sensory hair cells. After prolonged mechanical stimulation of hair cells, both phase locking of afferent nerve activity and the recovery of spontaneous release of synaptic vesicles are diminished in synj1 mutants. Presumably as a behavioral consequence of these synaptic deficits, synj1 mutants are unable to maintain an upright posture. To probe vestibular function with respect to postural control in synj1 mutants, we developed a method for assessing the vestibulospinal reflex (VSR) in larvae. We elicited the VSR by rotating the head and recorded tail movements. As expected, the VSR is completely absent in pcdh15a and lhfpl5a mutants that lack inner ear function. Conversely, lhfpl5b mutants, which have a selective loss of function of the lateral line organ, have normal VSRs, suggesting that the hair cells of this organ do not contribute to this reflex. In contrast to mechanotransduction mutants, the synj1 mutant produces normal tail movements during the initial cycles of rotation of the head. Both the amplitude and temporal aspects of the response are unchanged. However, after several rotations, the VSR in synj1 mutants was strongly diminished or absent. Mutant synj1 larvae are able to recover, but the time required for the reappearance of the VSR after prolonged stimulation is dramatically increased in synj1 mutants. Collectively, the data demonstrate a behavioral correlate of the synaptic defects caused by the loss of synj1 function. Our results suggest that defects in synaptic vesicle recycling give rise to fatigue of ribbons synapses and possibly other synapses of the VS circuit, leading to the loss of postural control.

Synaptojanin and endophilin represent a classic pair of endocytic proteins that exhibit coordinated action during rapid synaptic vesicle endocytosis. Current models suggest that synaptojanin activity is tightly associated with endophilin through high-affinity binding between the synaptojanin proline-rich domain (PRD) and the endophilin SH3 domain. Surprisingly, we find that truncated synaptojanin lacking the PRD domain sustains normal synaptic transmission, indicating that synaptojanin's core function in vivo resides in the remaining two domains that contain phosphoinositide-phosphatase activities: an N-terminal Sac1 phosphatase domain and a 5-phosphatase domain. We further show that the Sac1 domain plays an unexpected role in targeting synaptojanin to synapses. The requirement for Sac1 is bypassed by tethering the synaptojanin 5-phophatase to the endophilin membrane-bending Bin–Amphiphysin–Rvs (BAR) domain. Together, our results uncover an unexpected role for the Sac1 domain in vivo in supporting coincident action between synaptojanin and endophilin at synapses.

Synaptic vesicle exocytosis at chemical synapses is followed by compensatory endocytosis. Multiple pathways including Clathrin-mediated retrieval of single vesicles, bulk retrieval of large cisternae, and kiss-and-run retrieval have been reported to contribute to vesicle recycling. Particularly at the continuously active ribbon synapses of retinal photoreceptor and bipolar cells, compensatory endocytosis plays an essential role to provide ongoing vesicle supply. Yet, little is known about the mechanisms that contribute to endocytosis at these highly complex synapses. To identify possible specializations in ribbon synaptic endocytosis during different states of activity, we exposed mice to controlled lighting conditions and compared the distribution of endocytotic proteins at rod and cone photoreceptor, and ON bipolar cell ribbon synapses with light and electron microscopy. In mouse ON bipolar cell terminals, Clathrin-mediated endocytosis seemed to be the dominant mode of endocytosis at all adaptation states analyzed. In contrast, in mouse photoreceptor terminals in addition to Clathrin-coated pits, clusters of membranously connected electron-dense vesicles appeared during prolonged darkness. These clusters labeled for Dynamin3, Endophilin1, and Synaptojanin1, but not for AP180, Clathrin LC, and hsc70. We hypothesize that rod and cone photoreceptors possess an additional Clathrin-independent mode of vesicle retrieval supporting the continuous synaptic vesicle supply during prolonged high activity.