Potassium ion- and nitric oxide-induced exocytosis from populations of hippocampal synapses during synaptic maturation in vitro

doi:10.1016/S0306-4522(97)00152-8

Neuroscience

Volume 80, Issue 4, 11 August 1997, Pages 1057-1073

https://doi.org/10.1016/S0306-4522(97)00152-8 Get rights and content

Abstract

The development of mechanisms of neurotransmitter release is an important component in the formation of functional synaptic connections. Synaptic neurotransmitter release can be modulated by nitric oxide, a compound shown to have a variety of physiologic functions in the nervous system. The goal of this study was to determine whether, during synaptic maturation, nitric oxide is capable of affecting exocytosis of synaptic vesicles, and to compare its effects with those elicited by strongly depolarizing stimuli. To address these questions we examined vesicle release from large numbers of individual synapses of hippocampal neurons between five and 13 days in culture. Synaptic vesicles were labelled by uptake of the styrylpyridinium dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43) and their release was monitored by fluorescence imaging. Across populations of developing synapses, there was a good correspondence between FM1-43 staining and synapsin immunocytochemistry. A marked heterogeneity was observed in the ability to release vesicles both after potassium and nitric oxide stimulation. In less mature populations of synapses, the rate of potassium- and nitric oxide-induced exocytosis gradually increased, while at later stages nitric oxide-induced responses levelled off and potassium-induced responses continued to rise. Application of nitric oxide donors did not trigger any detectable changes in intracellular calcium. Combined immunocytochemical analysis of cultured hippocampal neurons for neuronal nitric oxide synthase and synapsin revealed that nitric oxide synthase was present within neurites of cultured hippocampal neurons, largely distributed in a bead-like pattern which partially overlapped presynaptic sites. Stimulation of the N-methyl-d-aspartate receptor while blocking propagation of action potentials with tetrodotoxin resulted in exocytosis from numerous individually resolved sites. Preincubation of neurons with an nitric oxide synthase inhibitor or addition of an nitric oxide scavenger eliminated these responses indicating a role for nitric oxide in N-methyl-d-aspartate-stimulated exocytosis.

Using fluorescence imaging of individually resolved synaptic sites, we provide direct evidence for an effect of nitric oxide on vesicular neurotransmitter release in intact neurons. Nitric oxide is capable to produce this effect at all stages of synaptic development and acts independently of calcium influx. We show that nitric oxide synthase is present at synaptic sites and endogenously produced nitric oxide is sufficient to cause exocytosis.

Taken together, these experiments suggest a possible role for nitric oxide in calcium-independent transmitter release in populations of synapses at all stages of maturation.

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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Potassium ion- and nitric oxide-induced exocytosis from populations of hippocampal synapses during synaptic maturation in vitro

Abstract

Section snippets

Cell culture

Responses of synaptic populations to potassium depolarization

Discussion

Conclusions

Acknowledgements

Cell

Neuron

Cell

Fedn Eur. biochem. Socs Lett.

Neuron

Neuron

Devl Brain Res.

Neurosci. Lett.

Neuron

Neuron

Neuron

Prog. Neurobiol.

Antagonistic action of imidazolineoxyl N-oxides against endothelium-derived relaxing factor/NO through a radical reaction

Biochemistry

An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture. II. Synaptic relationships

J. Neurosci.

Hippocampal synaptogenesis in cell culture: Developmental time course of synapse formation, calcium influx, and synaptic protein distribution

J. Neurosci.

Activity-dependent fluorescent staining and destaining of living motor nerve terminals

J. Neurosci.

Synaptic plasticity in rat hippocampal slice cultures: Local “Hebbian” conjunction of pre- and postsynaptic stimulation leads to distributed synaptic enhancement

Proc. natn. Acad. Sci. U.S.A.

Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination

J. Neurosci. Res.

A role for nitric oxide in the development of the ferret retinogeniculate projection

J. Neurosci.

The establishment of polarity by hippocampal neurons in culture

J. Neurosci.

Nitric oxide: Linking space and time in the brain

Proc. natn. Acad. Sci. U.S.A.

The biochemical pathways of nitric oxide formation from nitrovasodilators: Appropriate choice of exogenous NO donors and aspects of preparation and handling of aqueous NO solutions

J. cardiovasc. Pharmac.

The distribution of synapsin I and synaptophysin in hippocampal neurons developing in culture

J. Neurosci.

Synaptogenesis in hippocampal cultures: evidence indicating that axons and dendrites become competent to form synapses at different stages of neuronal development

J. Neurosci.

The NO hypothesis: Possible effects of a short-lived, rapidly diffusible signal in the development and function of the nervous system

Proc. natn. Acad. Sci. U.S.A.