Techniques: Applications of the nerve–bouton preparation in neuropharmacology

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Abstract

Single mammalian neurons can be isolated with adherent functional synaptic terminals using an enzyme-free, mechanical dissociation procedure. This allows investigations of the effects of presynaptic modulators of synaptic transmission with unprecedented ease and accuracy. Furthermore, single presynaptic terminals and boutons can be visualized using fluorescent markers and can also be focally stimulated with electrical pulses. In this article, the isolated-nerve–adherent-synaptic-bouton preparation and some examples of its general properties and uses are discussed.

Section snippets

History of the acutely isolated nerve–bouton preparation

The dissociation of neurons facilitates the ability to visualize and patch-clamp single neurons and to control the surrounding solutions. The use of acutely isolated neurons improves the ability to: (1) accurately measure the current kinetics and voltage dependence of these neurons (because the much reduced dendritic arbors often enables adequate space-clamp); and (2) ascribe the observed effect of a specific manipulation to the neuron under study, rather than to an indirect action mediated by

Adherent functional synaptic boutons

Electron micrographs and fluorescent imaging, using incorporation of either synaptophysin antibodies or FM1–43 into synaptic vesicles, reveals multiple small synaptic terminals, or boutons, adherent to the mechanically dissociated neurons (Fig. 1c,d). These boutons are functional, generating spontaneous synaptic currents with little decrement in amplitude or frequency for at least an hour after recordings commence 5, 6. The frequency of sIPSCs is typically much higher than for sEPSCs, which

General properties of spontaneous and evoked GABA release in the isolated nerve–bouton preparation

The frequency of sIPSCs recorded from different brain regions is typically 1.0–10.0 Hz; much of this variability presumably reflects differences in the excitability and number of adherent terminals. Both action-potential-dependent and -independent forms of GABA release contribute to sIPSCs, with tetrodotoxin (TTX) decreasing event frequency by 50–80% 4, 6. In the presence of TTX, sIPSC frequency can be enhanced by raising external K+ or can be further reduced (by 40–80%) by removal of

Neuropharmacological applications of the isolated nerve–bouton preparation

The ability to record frequent and sustained sIPSCs in well isolated and space-clamped neurons makes the preparation particularly suitable for studies of presynaptic receptor-induced modulation of glycine-mediated and GABA-mediated neurotransmission, and the underlying transduction mechanisms. Figure 3 describes a few selected examples of such studies. The metabotropic receptors shown inhibit GABA release by direct modulation of the release machinery, inhibition of presynaptic Ca2+ channels

Concluding remarks

The nerve–bouton preparation is a simple and additional approach to study neurotransmitter release from the vast majority of small-diameter mammalian nerve terminals. High rates of synaptic currents can be recorded in acute preparations, with accurate control of membrane voltage, cytoplasmic constituents and the solutions bathing single neurons. The preparation is devoid of complications arising from surrounding cells and from possible changes in protein distribution and/or function as a result

Acknowledgements

The work described in this review has been funded primarily by Grants-in-Aid for Scientific Research (No. 13307003) from the Ministry of Education, Science and Culture, Japan, the Japan Health Sciences Foundation (No. 21279, Research on Brain Science), and Kyushu University Interdisciplinary Programs in Education and Projects in Research Development (all to N.A.). A.J.M. acknowledges the support of an Australian Academy of Sciences/Japan Society for the Promotion of Science exchange program

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