Perfusion of Immobilized Isolated Nerve Terminals as a Model for the Regulation of Transmitter Release: Release of Different, Endogenous Transmitters, Repeated Stimulation, and High Time Resolution

Abstract

To study the release of neurotransmitters, i.e., the recruitment of transmitters for release and the regulation of the release process, isolated nerve terminals (synaptosomes) of the rat forebrain were immobilized in Sephadex gel inside a perfusion chamber. In this way, the following were achieved: (a) A very limited pressure stress was exerted on the synaptosomes, so that these remained viable for long periods (greater than 30 min) inside the chamber and did not elute from the chamber, which allowed long-term experiments with repeated stimulations; (b) estimation of the release of various endogenous transmitters, both in a Ca(2+)-dependent (exocytotic) and Ca(2+)-independent manner; (c) a step-like stimulation with depolarizing agents (rise time, 3-4 s) and a high time resolution (600-ms sampling); and (d) negligible reuptake of transmitter into the terminals or extracellular breakdown. It is concluded that this perfusion setup helps to provide new insights in the presynaptic stimulus-secretion coupling, co-transmission, and the exo-endocytosis cycle.

Differential signaling in presynaptic neurotransmitter release

“…Although LDCVs have been assumed to be unable for recycling and fully dependent on new synthesis of their peptide transmitter content in the cell body and subsequent transport towards the nerve terminal, recent evidence in neuroendocrine cells suggests that these larger vesicles regularly did not secrete their full peptide content during fusion and could be recycled in order to be used for a next exocytotic round [30,31]. Extrapolation of these findings to small nerve terminals requires care, but repeated secretion of peptide from isolated nerve terminals by subsequent massive stimulatory pulses would be in agreement with such a mechanism [32]. Besides the differential transmitter release from different vesicle types as a presynaptic instrument for functional diversification common in many synapses, the whole architecture of the nerve terminal is highly determining as well.…”

Regional differences in the effects of isoflurane on neurotransmitter release

“…It is likely that regional differences in anesthetic sensitivity also exist for other transmitters based on their distinct presynaptic regulatory mechanisms (Snyder et al, 2007; Verhage et al, 1992). This is exemplified by the more potent inhibition by isoflurane of acetylcholine-evoked norepinephrine release from hippocampal nerve terminals mediated by presynaptic nicotinic acetylcholine receptors (Westphalen et al, 2009), and by the lesser anesthetic sensitivity of neuropeptide transmitter release from large dense core vesicles (Pashkov and Hemmings, 2002).…”

α-Latrotoxin, Acting via Two Ca2+-dependent Pathways, Triggers Exocytosis of Two Pools of Synaptic Vesicles

“…For quick and thorough mixing and to prevent NT reuptake (20,25), synaptosomes (10 -25 g of protein) were diluted by the stimulus 8-fold. Using high K ϩ , maximal release was achieved at 25 mM K ϩ .…”