Kainate Modulates Presynaptic GABA Release from Two Vesicle Pools

Abstract

Inhibitory control of local neuronal circuits is critical for prefrontal cortical functioning. Modulation of inhibitory circuits by several neuromodulators has been demonstrated, but the underlying mechanisms are unclear. Neuromodulator effects on synaptic vesicle recycling have received little attention. Controversy also exists whether different pools of synaptic vesicles underlie spontaneous and activity-dependent vesicle recycling. We therefore investigated the effects of kainate receptor activation on GABA release in rat prefrontal neocortex using electrophysiological and styryl dye imaging techniques in acute neocortical slices. Electrophysiological studies demonstrated that activation of kainate receptors increased the frequency, but not the amplitude of miniature IPSCs, suggesting a presynaptic action. Using styryl dye staining and multiphoton excitation microscopy, we visualized vesicular release from inhibitory GABAergic terminals in prefrontal cortical slices and demonstrate that kainate facilitates GABA release from presynaptic terminals. Our findings also indicate the presence of two pools of GABA-containing vesicles within inhibitory terminals. Kainate modulates both pools but only when vesicles are endocytosed and exocytosed by matching protocols of dye loading, i.e., spontaneous or evoked afferent activity.

The same synaptic vesicles drive active and spontaneous release

“…While there still could be conditions that favor (in a limited way) some bias toward how synaptic vesicles form following one type of stimulation, and how they are later released, our results indicate that there is no strict segregation between vesicles used for evoked and spontaneous release. This conclusion disagrees with some of the previously published works 6,[8][9][10] . The source of this discrepancy is not completely clear, although pitfalls of the data analysis methods used in some of the previous studies have already been pointed to 7 .…”

“…Surprisingly, both of these findings have been later contested in similar experiments -FM dyes taken up either at rest or during stimulation could be released with identical kinetics, and the inhibition of reacidification during depolarization led to a cessation of spontaneous release 7 (see also 5 ). However, later studies have again found different FM dye release kinetics when using different loading paradigms 8,9 , and labeling experiments using the expression of a biotinylated variant of the synaptic vesicle marker VAMP2 (synaptobrevin) also supported the hypothesis that spontaneously and actively recycling vesicles are different 10 .We tested here this controversial issue by combining several fluorescence imaging assays. We sought to reproduce the FM dye loading/unloading paradigms used in the past.…”

“…We sought to reproduce the FM dye loading/unloading paradigms used in the past. A number of FM dyes have been used, including FM 1-43 and FM 2-10 6-9 ; both dyes have provided evidence for a difference between the spontaneously and actively recycling vesicles 8,9 . It has been recently suggested that FM 2-10 reports this difference better than FM 1-43 9 .…”

“…The systematic investigation of all possible loading/unloading paradigms (active-active, activespontaneous, spontaneous-active and spontaneous-spontaneous) had never been applied in past studies addressing the issue [6][7][8][9][10] . We tested four different synaptic preparations: the neuromuscular junctions (NMJs) of mouse, frog and Drosophila third instar larvae, cultured hippocampal neurons.…”

“…However, as most of the previous work [6][7][8][9][10] dealt with hippocampal cultures, we employed several additional experimental approaches in this model system. We first designed an assay in which the synaptic vesicle marker synaptotagmin was identified by biotinylated antibodies during one round of loading (actively for 30 seconds at 20 Hz, or spontaneously for 15 minutes).…”