Botulinum neurotoxin type A modulates vesicular release of glutamate from satellite glial cells

This study investigated the presence of cell membrane docking proteins synaptosomal‐associated protein, 25 and 23 kD (SNAP‐25 and SNAP‐23) in satellite glial cells (SGCs) of rat trigeminal ganglion; whether cultured SGCs would release glutamate in a time‐ and calcium‐dependent manner following calcium‐ionophore ionomycin stimulation; and if botulinum neurotoxin type A (BoNTA), in a dose‐dependent manner, could block or decrease vesicular release of glutamate. SGCs were isolated from the trigeminal ganglia (TG) of adult Wistar rats and cultured for 7 days. The presence of SNAPs in TG sections and isolated SGCs were investigated using immunohistochemistry and immunocytochemistry, respectively. SGCs were stimulated with ionomycin (5 μM for 4, 8, 12 and 30 min.) to release glutamate. SGCs were then pre‐incubated with BoNTA (24 hrs with 0.1, 1, 10 and 100 pM) to investigate if BoNTA could potentially block ionomycin‐stimulated glutamate release. Glutamate concentrations were measured by ELISA. SNAP‐25 and SNAP‐23 were present in SGCs in TG sections and in cultured SGCs. Ionomycin significantly increased glutamate release from cultured SGCs 30 min. following the treatment (P < 0.001). BoNTA (100 pM) significantly decreased glutamate release (P < 0.01). Results from this study demonstrated that SGCs, when stimulated with ionomycin, released glutamate that was inhibited by BoNTA, possibly through cleavage of SNAP‐25 and/or SNAP‐23. These novel findings demonstrate the existence of vesicular glutamate release from SGCs, which could potentially play a role in the trigeminal sensory transmission. In addition, interaction of BoNTA with non‐neuronal cells at the level of TG suggests a potential analgesic mechanism of action of BoNTA.     

Opposing functions of two sub‐domains of the SNARE‐complex in neurotransmission

The SNARE‐complex consisting of synaptobrevin‐2/VAMP‐2, SNAP‐25 and syntaxin‐1 is essential for evoked neurotransmission and also involved in spontaneous release. Here, we used cultured autaptic hippocampal neurons from Snap‐25 null mice rescued with mutants challenging the C‐terminal, N‐terminal and middle domains of the SNARE‐bundle to dissect out the involvement of these domains in neurotransmission. We report that the stabilities of two different sub‐domains of the SNARE‐bundle have opposing functions in setting the probability for both spontaneous and evoked neurotransmission. Destabilizing the C‐terminal end of the SNARE‐bundle abolishes spontaneous neurotransmitter release and reduces evoked release probability, indicating that the C‐terminal end promotes both modes of release. In contrast, destabilizing the middle or deleting the N‐terminal end of the SNARE‐bundle increases both spontaneous and evoked release probabilities. In both cases, spontaneous release was affected more than evoked neurotransmission. In addition, the N‐terminal deletion delays vesicle priming after a high‐frequency train. We propose that the stability of N‐terminal two‐thirds of the SNARE‐bundle has a function for vesicle priming and limiting spontaneous release.