Nobutoshi Harata

Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion

Exo–endocytotic turnover of synaptic vesicles (SVs) at synapses between hippocampal neurons in culture was examined by electron microscopy (EM). We carried out photoconversion (PC) of the fluorescent endocytotic marker FM 1-43 by using 3,3′-diaminobenzidine to convert the dye signal into an electron-dense product. Electron-dense products were located almost exclusively in SVs, whose densities were bimodally distributed in two sharply demarcated populations, PC-positive (PC+) and PC-negative (PC−). The median densities of these populations did not vary with the proportion of vesicles stained within a presynaptic terminal (bouton). The proportion of PC+ SVs remained constant across consecutive thin sections of single boutons, but varied greatly from one bouton to another, indicating marked heterogeneity in exo-endocytotic activity. Our experiments indicated that only a minority of SVs were stained in most boutons after stimuli known to cause complete turnover of the functional vesicular pool. A direct spatial correlation was found between FM 1-43 fluorescent spots seen with light microscopy and PC+ boutons by EM. The correlation was clearer in isolated boutons than in clusters of boutons. Photoconversion in combination with FM dyes allows clarification of important aspects of vesicular traffic in central nervous system nerve terminals.

Limited numbers of recycling vesicles in small CNS nerve terminals: implications for neural signaling and vesicular cycling

The tiny nerve terminals of central synapses contain far fewer vesicles than preparations commonly used for analysis of neurosecretion. Photoconversion of vesicles rendered fluorescent with the dye FM1-43 directly identified vesicles capable of engaging in exo-endocytotic recycling following stimulated Ca(2+) entry. This recycling pool typically contained 30-45 vesicles, only a minority fraction (15-20% on average) of the total vesicle population. The smallness of the recycling pool would severely constrain rates of quantal neurotransmission if classical pathways were solely responsible for vesicle recycling. Fortunately, vesicles can undergo rapid retrieval and reuse in addition to conventional slow recycling, to the benefit of synaptic information flow and neuronal signaling.

Kiss-and-run and full-collapse fusion as modes of exo-endocytosis in neurosecretion.

Neurotransmitters and hormones are released from neurosecretory cells by exocytosis (fusion) of synaptic vesicles, large dense‐core vesicles and other types of vesicles or granules. The exocytosis is terminated and followed by endocytosis (retrieval). More than fifty years of research have established full‐collapse fusion and clathrin‐mediated endocytosis as essential modes of exo‐endocytosis. Kiss‐and‐run and vesicle reuse represent alternative modes, but their prevalence and importance have yet to be elucidated, especially in neurons of the mammalian CNS. Here we examine various modes of exo‐endocytosis across a wide range of neurosecretory systems. Full‐collapse fusion and kiss‐and‐run coexist in many systems and play active roles in exocytotic events. In small nerve terminals of CNS, kiss‐and‐run has an additional role of enabling nerve terminals to conserve scarce vesicular resources and respond to high‐frequency inputs. Full‐collapse fusion and kiss‐and‐run will each contribute to maintaining cellular communication over a wide range of frequencies.

Frequency-Dependent Kinetics and Prevalence of Kiss-and-Run and Reuse at Hippocampal Synapses Studied with Novel Quenching Methods

The kinetics of exo-endocytotic recycling could restrict information transfer at central synapses if neurotransmission were entirely reliant on classical full-collapse fusion. Nonclassical fusion retrieval by kiss-and-run would be kinetically advantageous but remains controversial. We used a hydrophilic quencher, bromophenol blue (BPB), to help detect nonclassical events. Upon stimulation, extracellular BPB entered synaptic vesicles and quenched FM1-43 fluorescence, indicating retention of FM dye beyond first fusion. BPB also quenched fluorescence of VAMP (synaptobrevin-2)-EGFP, thus indicating the timing of first fusion of vesicles in the total recycling pool. Comparison with FM dye destaining revealed that kiss-and-run strongly prevailed over full-collapse fusion at low frequency, giving way to a near-even balance at high frequency. Quickening of kiss-and-run vesicle reuse was also observed at higher frequency in the average single vesicle fluorescence response. Kiss-and-run and reuse could enable hippocampal nerve terminals to conserve scarce vesicular resources when responding to widely varying input patterns.

Recordings from Single Neocortical Nerve Terminals Reveal a Nonselective Cation Channel Activated by Decreases in Extracellular Calcium

Synaptic activity causes reductions in cleft [Ca(2+)] that may impact subsequent synaptic efficacy. Using modified patch-clamp techniques to record from single neocortical nerve terminals, we report that physiologically relevant reductions of extracellular [Ca(2+)] ([Ca(2+)](o)) activate voltage-dependent outward currents. These outward currents are carried by a novel nonselective cation (NSC) channel that is indirectly inhibited by various extracellular agents (rank order potency, Gd(3+) > spermidine > Ca(2+) > Mg(2+), typical for [Ca(2+)](o) receptors). The identification of a Ca(2+) sensor-NSC channel pathway establishes the existence of a mechanism by which presynaptic terminals can detect and respond to reductions in cleft [Ca(2+)]. Activation of NSC channels by falls in [Ca(2+)](o) would be expected during periods of high activity in the neocortex and may modulate the excitability of the presynaptic terminal.

Gliclazide Attenuates the Intracellular Ca2+ Changes Induced In Vitro by Ischemia in the Retinal Slices of Rats with Streptozotocin-Induced Diabetes

Purpose: To investigate the dynamics of the intracellular Ca2+ concentration ([Ca2+]i) during retinal ischemia in rats with streptozotocin (STZ)-induced diabetes and the effect of gliclazide, a sulfonylurea with a potent free-radical scavenging activity on ischemia-induced [Ca2+]i dynamics. Methods: Rats with STZ (65 mg/kg) induced diabetes were divided into three groups: the untreated diabetic group, the gliclazide-treated group, and the glibenclamide-treated group. An ischemic condition was imposed in vitro on the retinal slices by perfusion with an oxygen/glucose deprived solution. The [Ca2+]i was measured in individual layers of the rat retinal slices loaded with the Ca2+ indicator fluo-3. Results: As compared to that in the normal rat retina, both the amplitude and the kinetics of the [Ca2+]i increase were suppressed in the intermediate layers of the retinal slices from the diabetic rats under the ischemic condition. These changes were attenuated by the administration of gliclazide but not by that of glibenclamide. Conclusions: Hyperglycemia influences ischemia-induced [Ca2+]i dynamics predominantly in the intermediate layers of the retina, and gliclazide, as compared to glibenclamide without a free radical scavenging activity, potently attenuates the ischemia-induced changes in the calcium dynamics.