Potassium ion- and nitric oxide-induced exocytosis from populations of hippocampal synapses during synaptic maturation in vitro
Section snippets
Cell culture
All experiments have been carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Neurosciences Institute and with federal and state policies; in particular all efforts were made to minimize animal suffering and to reduce the number of animals used. Primary cultures of hippocampal neurons were prepared from E19 embryonic rats (Wistar Kyoto, Harlan). In brief, hippocampi were dissected and connective tissue removed. The pooled tissue was
Responses of synaptic populations to potassium depolarization
To adequately compare different experimental conditions and developmental stages we monitored responses from large numbers of individually resolved synaptic sites. This revealed significant heterogeneity among synaptic responses for a given stimulation period. After staining with FM1-43 in the presence of 90 mM K+ and subsequent wash-out (5–10 min) of excess dye, neurons (10 DIV) showed numerous fluorescent spots or punctae along their neurites (Fig. 1A,B). A second depolarizing stimulus (30 s, 90
Discussion
The primary goal of this study was to determine whether NO is capable of triggering exocytosis from developing hippocampal synapses and how significant the effects of NO might be with respect to “classical” calcium-dependent mechanisms. This analysis and comparison required the quantitation of large numbers of responses obtained from individually resolved synaptic sites. Using FM1-43 fluorescence imaging we performed a population analysis of exocytosis at different stages of development in vitro
Conclusions
In summary, our experiments demonstrate that nitric oxide is capable of triggering exocytosis of synaptic vesicles during synaptic maturation in hippocampal culture. Fluorescence imaging of individually-resolved synaptic sites indicates that the degree of exocytosis following stimulation is highly variable within any given developing synaptic population, and that nitric oxide-induced exocytosis occurs without a detectable rise in intracellular calcium. Stimulation of the NMDA receptor results
Acknowledgements
This work was supported by Neurosciences Research Foundation. The Foundation receives major support from Sandoz Pharmaceutical Corporation.
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