Superfusion of synaptosomes to study presynaptic mechanisms involved in neurotransmitter release from rat brain

Abstract

Neurotransmitter release, as the primary way for neuron signaling, represents the target of a staggering number of studies in order to understand complex neural functions. The corpus striatum is a brain area especially rich in neurotransmitters where cholinergic neurons are supposed to play an associative role between different neuronal types, and therefore their activity is modulated by multiple neurotransmitter systems [Trends Neurosci. 17 (1994) 228; Trends Neurosci. 18 (1995) 527] [13,25]. In this regard, superfusion of synaptosomes is a useful in vitro approach to study the neurotransmitter release allowing an unequivocal interpretation of results obtained under accurately specified experimental conditions. Synaptosomes are sealed presynaptic nerve terminals obtained after homogenating brain tissue in iso-osmotic conditions [J. Physiol. 142 (1958) 187] [22]. Synaptosomes have been extensively used to study the mechanism of neurotransmitter release in vitro because they preserve the biochemical, morphological and electrophysiological properties of the synapse [J. Neurocytol. 22 (1993) 735] [42]. The superfusion, strictly a perfusion, allows both the continuous removal of the compounds present in the biophase of the presynaptic proteins and the easy exchange of the medium. We herein describe the method of superfusion of rat striatal synaptosomes to study the [3H]ACh release under basal and stimulated conditions. To depolarize the synaptosomal preparation three different strategies were employed: high extracellular concentration of K+ (15 mM), a K+ channel-blocker (4-aminopyridine, 1–30 μM), or veratridine (10 μM) which blocks the inactivation of voltage-dependent Na+ channels.