In synapses, a rise in presynaptic intracellular calcium leads to secretory vesicle fusion in less than a millisecond, as indicated by the short delay from excitation to postsynaptic signal. In nonsynaptic secretory cells, studies at high time resolution have been limited by the lack of a detector as fast and sensitive as the postsynaptic membrane. Electrochemical methods may be sensitive enough to detect catecholamines released from single vesicles. Here, we show that under voltage-clamp conditions, stochastically occurring signals can be recorded from adrenal chromaffin cells using a carbon-fibre electrode as an electrochemical detector. These signals obey statistics characteristic for quantal release; however, in contrast to neuronal transmitter release, secretion occurs with a significant delay after short step depolarizations. Furthermore, we identify a pedestal or 'foot' at the onset of unitary events which may represent the slow leak of catecholamine molecules out of a narrow 'fusion pore' before the pore dilates for complete exocytosis.

The kinetics of the secretory response in bovine chromaffin cells following flash photolysis of caged Ca2+ were studied by capacitance (Cm) measurements with millisecond time resolution. After elevation of the internal Ca2+ concentration ([Ca2+]i), Cm rises rapidly with one or more exponentials. The time constant of the fastest component decreases for higher [Ca2+]i (range 3-600 microM) over three orders of magnitude before it saturates at approximately 1 ms. The corresponding maximal rates of secretion can be as fast as 100,000 fF/s or 40,000 vesicles/s. There is a Ca(2+)-dependent delay before Cm rises, which may reflect the kinetics of multiple Ca2+ ions binding to the secretory apparatus. The initial rise in Cm is described by models containing a sequence of two to four single Ca(2+)-binding steps followed by a rate-limiting exocytosis step. The predicted Ca2+ dissociation constant (Kd) of a single Ca(2+)-binding site is between 7 and 21 microM. At [Ca2+]i > 30 microM clear indications of a fast endocytotic process complicate the analysis of the secretory response.

Retinal prosthetic implants are the only approved treatment for retinitis pigmentosa, a disease of the eye that causes blindness through gradual degeneration of photoreceptors. An array of microelectrodes triggered by input from a camera stimulates surviving retinal neurons, each electrode acting as a pixel. Unintended stimulation of retinal ganglion cell axons causes patients to see large, oblong shapes of light, rather than focal spots, making it difficult for them to perceive forms. To address this problem, we performed calcium imaging in isolated retinas and mapped the patterns of cells activated by different electrical stimulation protocols. We found that pulse durations two orders of magnitude longer than those typically used in existing implants stimulate inner retinal neurons while avoiding activation of ganglion cell axons, thus confining retinal responses to the site of the electrode. We show that multielectrode stimulation with 25-ms pulses can pattern letters on the retina corresponding to a Snellen acuity of 20/312. We validated our findings in a patient with an implanted epiretinal prosthesis by demonstrating that 25-ms pulses evoke focal spots of light.

Synaptotagmin I is a synaptic vesicle-associated protein essential for synchronous neurotransmission. We investigated its impact on the intracellular Ca 2؉ -dependence of large dense-core vesicle (LDCV) exocytosis by combining Ca 2؉ -uncaging and membrane capacitance measurements in adrenal slices from mouse synaptotagmin I null mutants. Synaptotagmin I-deficient chromaffin cells displayed prolonged exocytic delays and slow, yet Ca 2؉ -dependent fusion rates, resulting in strongly reduced LDCV release in response to short depolarizations. Vesicle recruitment, the shape of individual amperometric events, and endocytosis appeared unaffected. These findings demonstrate that synaptotagmin I is required for rapid, highly Ca 2؉ -sensitive LDCV exocytosis and indicate that it regulates the equilibrium between a slowly releasable and a readily releasable state of the fusion machinery. Alternatively, synaptotagmin I could function as calcium sensor for the readily releasable pool, leading to the destabilization of the pool in its absence.T he release of neurotransmitters from nerve terminals and hormones from neuroendocrine cells occurs through exocytosis of secretory vesicles in response to increases in the intracellular Ca 2ϩ concentration [Ca 2ϩ ] i (1). The supralinear Ca 2ϩ dependence of neurosecretion suggests that the binding of at least 3-5 Ca 2ϩ ions to Ca 2ϩ -sensing entities on the fusion machinery is required to trigger the rapid fusion of secretory vesicle with the plasma membrane (2-6). At present, the exact mechanism of Ca 2ϩ -dependent exocytosis and the molecular identity of the involved Ca 2ϩ sensor(s) remain matters of debate. Numerous studies indicate that the synaptic vesicle protein synaptotagmin I, a brain-enriched member of the synaptotagmin family, plays a key role in Ca 2ϩ -dependent neurosecretion. Synaptotagmin I has been described to interact with several synaptic proteins including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxin (7) and SNAP-25 (8), the assembled SNARE complex (9-11), and the clathrin assembly protein complex AP-2 (12). Through the first of its two C 2 domains (C 2 A), synaptotagmin I binds Ca 2ϩ and rapidly interacts with phospolipid membranes in a Ca 2ϩ -dependent manner (9, 13). Functional evidence has been presented that implicate synaptotagmin I in vesicle docking (14), fusion (15)(16)(17)(18)(19)(20), and recycling (21). Most strikingly, gene mutation studies in mice (20,22) demonstrated that synaptotagmin I is specifically required for rapid synchronous neurotransmission but not for asynchronous or Ca 2ϩ -independent release (i.e., spontaneous release and release triggered by hypertonic solutions or ␣-latrotoxin). This finding led to the hypothesis that synaptotagmin I is the major Ca sensor, whereas the kinetics of exocytosis were simultaneously determined by using high time-resolution membrane capacitance (C m ) measurements. Using these methods, we demonstrate that synaptotagmin I-deficient chromaffin cells d...

Recent experiments on a variety of neuroendocrine cells indicate that intense stimuli readily depress the secretory response. The most likely explanation for this depression is that a pool of release-ready granules is depleted. We present a two-step model of secretion that allows one to simulate the dynamics of such a pool for different time courses of free intracellular Ca concentration [Ca2+]i. We derive rate constants of the model from two types of experiment and find that, for the simplest type of model, not only the rate of consumption (exocytosis) but also the rate of vesicle supply to the pool of release-ready granules must be made Ca-dependent. Given these functional dependences a variety of results from the literature can be simulated. In particular, the model predicts the occurrence of secretory depression and augmentation under appropriate conditions.

Synaptic terminals and neuroendocrine cells are packed with secretory vesicles, only a few of which are docked at the plasma membrane and readily releasable. The remainder are thought to constitute a large cytoplasmic reserve pool awaiting recruitment into the readily releasable pool (RRP) for exocytosis. How vesicles are prioritized in recruitment is still unknown: the choice could be random, or else the oldest or the newest ones might be favoured. Here we show, using a fluorescent cargo protein that changes colour with time, that vesicles in bovine adrenal chromaffin cells segregate into distinct populations, based on age. Newly assembled vesicles are immobile (morphologically docked) at the plasma membrane shortly after biogenesis, whereas older vesicles are mobile and located deeper in the cell. Different secretagogues selectively release vesicles from the RRP or, surprisingly, selectively from the deeper cytoplasmic pool. Thus, far from being equal, vesicles are segregated functionally and spatially according to age.

Single-vesicle release of catecholamines from chromaffin cells can be detected in real time as current spikes by the electrochemical method of amperometry. About 70% of spikes are preceded by a small "foot," the trickle of transmitter out of the early fusion pore. In addition, 20-50% of foot signals exhibit rapid fluctuations that we interpret as flickering of the fusion pore. There are also "stand-alone" foot signals, which may reflect transient fusions, in which the vesicles do not collapse completely into the plasma membrane. The number and frequency of the foot flickering are affected by intracellular Ca2+ concentration.

Substantia nigra neurons release dopamine from their somatodendritic regions. A long-unresolved question is whether this release occurs by exocytosis or by a nonvesicular mechanism. We used carbon fiber microelectrodes in a brainstem slice to assay secretion from single cell bodies that had been cleared of connective tissue. Amperometry at the carbon fiber microelectrodes revealed unitary events in approximately 90% of cells in resting conditions. These events had charge integrals ranging from a few femtocoulombs to several hundred femtocoulombs (fC). Local glutamate application enhanced the event frequency by 3.5-fold on average and up to 10-fold in highly responsive cells, although the mean charge integral was not modified. Local application of a high K+-containing saline had effects similar to those of glutamate. The frequency of resting and stimulated amperometric events was much lower at 21-22 degreesC than at 32-35 degreesC. The addition of Cd2+ (50 microM), a blocker of voltage-dependent Ca2+ channels, to the bath solution blocked the stimulatory effects of glutamate. These results suggest that dopamine is released from the somata of substantia nigra neurons by exocytosis and that this mechanism is regulated by neuronal electrical activity. More generally, this study demonstrates the applicability of carbon fiber microelectrodes to the measurement of quantal monoamine secretion in brain slices.