EPSCs at the synapses of sensory receptors and of some CNS neurons include large events thought to represent the synchronous release of the neurotransmitter contained in several synaptic vesicles by a process known as multiquantal release. However, determination of the unitary, quantal size underlying such putatively multiquantal events has proven difficult at hair cell synapses, hindering confirmation that large EPSCs are in fact multiquantal. Here, we address this issue by performing presynaptic membrane capacitance measurements together with paired recordings at the ribbon synapses of adult hair cells. These simultaneous presynaptic and postsynaptic assays of exocytosis, together with electron microscopic estimates of single vesicle capacitance, allow us to estimate a single vesicle EPSC charge of approximately Ϫ45 fC, a value in close agreement with the mean postsynaptic charge transfer of uniformly small EPSCs recorded during periods of presynaptic hyperpolarization. By thus establishing the magnitude of the fundamental quantal event at this peripheral sensory synapse, we provide evidence that the majority of spontaneous and evoked EPSCs are multiquantal. Furthermore, we show that the prevalence of uniquantal versus multiquantal events is Ca 2ϩ dependent. Paired recordings also reveal a tight correlation between membrane capacitance increase and evoked EPSC charge, indicating that glutamate release during prolonged hair cell depolarization does not significantly saturate or desensitize postsynaptic AMPA receptors. We propose that the large EPSCs reflect the highly synchronized release of multiple vesicles at single presynaptic ribbon-type active zones through a compound or coordinated vesicle fusion mechanism.

The proper wiring of the vertebrate brain represents an extraordinary developmental challenge, requiring billions of neurons to select their appropriate synaptic targets. In view of this complexity, simple vertebrate systems provide necessary models for understanding how synaptic specificity arises. The posterior lateral-line organ of larval zebrafish consists of polarized hair cells organized in discrete clusters known as neuromasts. Here we show that each afferent neuron of the posterior lateral line establishes specific contacts with hair cells of the same hair-bundle polarity. We quantify this specificity by modeling the neuron as a biased selector of hair-cell polarity and find evidence for bias from as early as 2.5 d after fertilization. More than half of the neurons form contacts on multiple neuromasts, but the innervated organs are spatially consecutive and the polarity preference is consistent. Using a novel reagent for correlative electron microscopy, HRP-mCherry, we show that these contacts are indeed afferent synapses bearing vesicle-loaded synaptic ribbons. Moreover, afferent neurons reassume their biased innervation pattern after hair-cell ablation and regeneration. By documenting specificity in the pattern of neuronal connectivity during development and in the context of organ regeneration, these results establish the posterior lateral-line organ as a vertebrate system for the in vivo study of synaptic target selection.

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The development of functional neural circuits requires that connections between neurons be established in a precise manner. The mechanisms by which complex nervous systems perform this daunting task remain largely unknown. In the posterior lateral line of larval zebrafish, each afferent neuron forms synaptic contacts with hair cells of a common hair-bundle polarity. We investigated whether afferent neurons distinguish hair-cell polarities by analyzing differences in the synaptic signaling between oppositely polarized hair cells. By examining two mutant zebrafish lines with defects in mechanoelectrical transduction, and by blocking transduction during the development of wild-type fish, we found that afferent neurons could form specific synapses in the absence of stimulus-evoked patterns of synaptic release. Asking next whether this specificity arises through intrinsically generated patterns of synaptic release, we found that the polarity preference persisted in two mutant lines lacking essential synaptic proteins. These results indicate that lateral-line afferent neurons do not require synaptic activity to distinguish hair-cell polarities and suggest that molecular labels of hair-cell polarity guide prepatterned afferents to form the appropriate synapses.calcium channel ͉ hair cell ͉ neuromast ͉ planar cell polarity ͉ protocadherin A n essential feature of neural development is the establishment of specific synaptic connections. To form the appropriate contacts, each growing axon must respond to guidance cues, find its target region, and then establish synapses with specific target cells (1, 2). The first two of these steps-axonal guidance and target recognition-rely predominantly on molecular signposts that attract or repulse growth cones in a manner independent of neuronal activity (3, 4). How neurons decide to form stable synapses with particular target cells, however, remains unclear. Activity serves an important role in regulating the growth of axonal arbors and in selectively stabilizing synapses (5-8). In several vertebrate systems, axons form synapses diffusely within the target region and then undergo activitydependent pruning to eliminate inappropriate synapses (9-14). Hebb's postulate, by which correlated activity between synaptic partners strengthens connections (15,16), offers an attractive model to explain this phenomenon (17). Nevertheless, the evidence for an activity-dependent process must be reconciled with data suggesting that normal brain architecture can form in the absence of synaptic transmission (18)(19)(20). In this case, synaptic specificity could derive from a combinatorial code of cell-surface molecules such as cadherins (21) or members of the immunoglobin superfamily (22). These fundamental uncertainties highlight the need for in vivo studies in an experimentally tractable vertebrate system.The posterior lateral line of zebrafish permits a detailed analysis of the role of activity in establishing synaptic specificity. The larval posterior lateral line consists of superficial clusters ...