Ultrastructural characterization of peri-synaptic astrocytic processes around cerebellar Purkinje spines under resting and stimulated conditions.

Abstract

In mammalian brains, astroglia presence near glutamatergic excitatory synapses has generated the term "tripartite" junctions, based on the close association of astrocytic processes near the active zone formed by presynaptic axonal terminal and postsynaptic dendritic spines. One major function of these astrocytic processes is to take up glutamate that spill out of the synaptic cleft during activity, via glutamate transporters located on astroglial plasma membrane. Comapred to other regions of the brain, the cerebellar Purkinje spines in the molecular layer are virtually completely ensheathed by Bergman glia, a special type of astrocyte, unique to cerebellum. The present electron microscopy study classifies these peri-synaptic astrocytic processes (PAP) ensheathing the Purkinje spine synapses into three types based on structural criteria: (1) Type 1- astrocytic process is situated at the edge of the synaptic cleft immediately next to the synaptic active zone. Under fast perfusion fixation conditions where synapses were under resting states, ~ 58% of the PAP's were scored as Type 1. The occurrence frequency of Type 1 PAP significantly decreased to 25% upon a 5-8 min delay in perfusion fixation, where synapses were under stimulated states. (2) Type 2- astrocytic process covers part of the postsynaptic membrane containing the postsynaptic density (PSD), so that this part of the PSD is separated from its presynaptic terminal. Occurrence frequency of Type 2 PAP's significantly increased from ~ 14% under fast perfusion fixation to 31% upon delayed perfusion fixation, and the average length of the PSD edge covered by astroglia increased from 41 nm to 57 nm upon delayed perfusion fixation. (3) Type 3- astrocytic process is situated some distance away from the active zone, while the presynaptic axon terminal extends to enwrap the spine beyond the active zone. Occurrence frequency of Type 3 PAP's increased from 28 to 43% upon delayed perfusion fixation, and the average length between apposed axon terminal and spine beyond the synaptic cleft significantly increased from 98 to 209 nm upon delayed perfusion fixation. Thus, upon stimulation, the tripartite synaptic junctions undergo dynamic structural changes with the astrocytic processes moving into the open cleft to cover the exposed postsynaptic membrane containing PSD, the presynaptic axon terminals extending to wrap the postsynaptic spine beyond the synaptic cleft. Both structural changes may facilitate glutamate uptake to clear the transmitter spilled out from the synaptic cleft during intense activity and prevent damage from overstimulation.