Ribbon synapses of the vertebrate retina are specialized synapses that release neurotransmitter by synaptic vesicle exocytosis in a manner that is proportional to the level of depolarization of the cell. This release property is different from conventional neurons, in which the release of neurotransmitter occurs as a short-lived burst triggered by an action potential. Synaptic vesicle exocytosis is a calcium regulated process that is dependent on a set of interacting synaptic proteins that form the so-called SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex. Syntaxin 3B has been identified as a specialized SNARE molecule in ribbon synapses of the rodent retina. However, the best physiologically-characterized neuron that forms ribbon-style synapses is the rod-dominant or Mb1 bipolar cell of the goldfish retina. We report here the molecular characterization of syntaxin 3B from the goldfish retina. Using a combination of reverse transcription (RT) PCR and immunostaining with a specific antibody, we show that syntaxin 3B is highly enriched in the plasma membrane of bipolar cell synaptic terminals of the goldfish retina. Using membrane capacitance measurements we demonstrate that a peptide derived from goldfish syntaxin 3B inhibits synaptic vesicle exocytosis. These experiments demonstrate that syntaxin 3B is an important factor for synaptic vesicle exocytosis in ribbon synapses of the vertebrate retina.

Mast cell degranulation is a highly regulated, calciumdependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis. Mast cells (MCs)2 participate in adaptive and innate immune responses. Their secreted products play important roles in immunoglobulin E (IgE)-dependent inflammatory reactions such as allergic asthma and anaphylaxis (1) and are also involved in other forms of inflammation such as immune arthritis (2, 3) and innate immune responses to bacterial infections (4, 5). Upon activation, MCs exhibit three main secretory responses: release of granule contents (i.e. degranulation), secretion of prostaglandins and leukotrienes, and secretion of cytokines and growth factors (6). The exocytic release of preformed mediators (e.g. histamine and proteases) stored in secretory granules is immediate and regulated at the step of fusion between the membrane of the granule and the plasma membrane. Thus, it is an example of regulated exocytosis, like neuronal synaptic neurotransmitter release and insulin secretion (7). Another early event is the release of metabolites of arachidonic acid (e.g. prostaglandin D 2 (PGD 2 ) and leukotriene C 4 (LTC 4 )). These eicosanoids cross the plasma membrane using transmembrane transporters (8), and their production is regulated by the activation of their synthetic enzymes (9). A late response after MC activation is the secretion of cytokines and growth factors (e.g. tumor necrosis factor-␣ (TNF-␣) and interleukin-4 (IL-4)). The gap in time of minutes to hours between stimulation and the secretion of these mediators is explained by the fact that regulation is at the transcriptional and post-transcriptional levels, with secretion occurring via constitutive exocytosis (10).A common intracellular mediator linking the stimulation event to these three MC responses is calcium (Ca 2ϩ ) that is released into the cytoplasm from intracellular stores and introduced from the extracellular environment via specialized channels. Increase in the cytoplasmic concentration of Ca 2ϩ ([Ca 2ϩ ] i ) is required for the activation of phospholi...

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Histamine released from retinopetal axons in the mouse retina can elevate intracellular calcium levels in the perikarya of dopaminergic cells via the activation of histamine H(1)-receptors.

Neurons that form ribbon-style synapses are specialized for continuous exocytosis. To this end, their synaptic terminals contain numerous synaptic vesicles, some of which are ribbon associated, that have difference susceptibilities for undergoing Ca-dependent exocytosis. In this study, we probed the relationship between previously defined vesicle populations and determined their fusion competency with respect to SNARE complex formation. We found that both the rapidly releasing vesicle pool and the releasable vesicle pool of the retinal bipolar cell are situated at the ribbon-style active zones, where they functionally interact. A peptide inhibitor of SNARE complex formation failed to block exocytosis from either pool, suggesting that these two vesicle pools have formed the SNARE complexes necessary for fusion. By contrast, a third, slower component of exocytosis was blocked by the peptide, as was the functional replenishment of vesicle pools, indicating that few vesicles outside of the ribbon-style active zones were initially fusion competent. In cone photoreceptors, similar to bipolar cells, fusion of the initial ribbon-associated synaptic vesicle cohort was not blocked by the SNARE complex-inhibiting peptide, whereas a later phase of exocytosis, attributable to the recruitment and subsequent fusion of vesicles newly arrived at the synaptic ribbons, was blocked. Together, our results support a model in which stimulus-evoked exocytosis in retinal ribbon synapses is SNARE-dependent; where vesicles higher up on the synaptic ribbon replenish the rapidly releasing vesicle pool; and at any given time, there are sufficient SNARE complexes to support the fusion of the entire ribbon-associated cohort of vesicles. An important implication of these results is that ribbon-associated vesicles can form intervesicular SNARE complexes, providing mechanistic insight into compound fusion at ribbon-style synapses.