Distribution of vesicle pools in cerebellar parallel fibre terminals after depression of ectopic transmission

At parallel fibre terminals in the cerebellar cortex, glutamate released outside of the active zone can activate AMPA receptors on juxtaposed Bergmann glial cell processes. This process is termed "ectopic" release, and allows for directed transmission to astroglial cells that is functionally independent of synaptic transmission to postsynaptic Purkinje neurons. The location of ectopic sites in presynaptic terminals is uncertain. Functional evidence suggests that stimulation of parallel fibres at 1 Hz exhausts ectopic transmission due to a failure to rapidly recycle vesicles to the ectopic pool, and so would predict a loss of vesicles in the near vicinity of extrasynaptic glial processes. In this study we used this stimulation protocol to investigate whether the distribution of vesicles within the presynaptic terminal is altered after suppression of ectopic release. Stimulation at 1 Hz had only a minor impact on the distribution of vesicles in presynaptic terminals when analysed with electron microscopy. Vesicle number and terminal size were unaffected by 1 Hz stimulation, but the relative abundance of vesicles in close proximity to the active zone was marginally reduced. In contrast, the fraction of vesicles facing glial membranes was unchanged after suppression of ectopic transmission. 1 Hz stimulation also resulted in a small but statistically-significant increase in the distance between glial membrane and presynaptic terminal, suggesting withdrawal of glial membranes from synapses is detectable in ultrastructural anatomy within minutes. These results raise doubts about the location of ectopic release sites, but indicate that neuron-glial association varies on a dynamic time scale.