Rapid turnover of spinules at synaptic terminals

Spinules found in brain consist of small invaginations of plasma membranes which enclose membrane evaginations from adjacent cells. Here, we focus on the dynamic properties of the most common type, synaptic spinules, which reside in synaptic terminals. In order to test whether depolarization triggers synaptic spinule formation, hippocampal slice cultures (7-day-old rats, 10-14 days in culture) were exposed to high K⁺ for 0.5-5 min, and examined by electron microscopy. Virtually no synaptic spinules were found in control slices representing a basal state, but numerous spinules appeared at both excitatory and inhibitory synapses after treatment with high K⁺. Spinule formation peaked with ∼1 min treatment at 37 °C, decreased with prolonged treatment, and disappeared after 1-2 min of washout in normal medium. The rate of disappearance of spinules was substantially slower at 4 °C. N-methyl-d-aspartic acid (NMDA) treatment also induced synaptic spinule formation, but to a lesser extent than high K⁺ depolarization. In acute brain slices prepared from adult mice, synaptic spinules were abundant immediately after dissection at 4 °C, extremely rare in slices allowed to recover at 28 °C, but frequent after high K⁺ depolarization. High pressure freezing of acute brain slices followed by freeze-substitution demonstrated that synaptic spinules are not induced by chemical fixation. These results indicate that spinules are absent in synapses at low levels of activity, but form and disappear quickly during sustained synaptic activity. The rapid turnover of synaptic spinules may represent an aspect of membrane retrieval during synaptic activity.