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|Title:||ACTIVITY-DEPENDENT MODULATION OF ASTROCYTIC STRUCTURES IN AREA CA1 OF MATURE HIPPOCAMPUS|
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|Authors/Affiliations:||2 David Verbich*; 1 Michael HaberDepartment of Neurology a; 1 Keith Murai; 3 R. Anne McKinney; |
1 Department of Neurology and Neurosurgery, Centre for Research in Neuroscience, McGill University, Montreal, QC, Canada; 2 Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada; 3 Department of Pharmacology and Therapeutics, Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada
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|Content:||Objectives: The hippocampus, an area of the brain important for learning and memory, exhibits long-lasting activity-dependent modulation of synaptic efficacy associated with both biochemical and structural changes. Approximately 57% of CA1 hippocampal synapses are closely apposed by astrocytic processes. Recent time-lapse imaging studies have shown that dendritic spines, the postsynaptic recipients of most excitatory transmission, and their associated astrocytic processes are highly dynamic, undergoing structural modifications over the time scale of minutes. These modifications are hypothesized to follow the activity of synapses and modulate neurotransmission. This study investigates astrocyte dynamics during alterations of synaptic activity in area CA1 of mature mouse hippocampus.|
Materials and Methods: We used 4D confocal imaging to investigate dynamics of protoplasmic CA1 astrocytes. Organotypic hippocampal slice cultures derived from P6 mice were maintained in culture for ≥ 21 days prior to transfection with Semliki Forest virus (SFV) strain A7 (74) to specifically label glial cells. This strain expresses membrane-targeted farnesylated mCherry permitting us to visualize mature, protoplasmic astrocytes with complex morphologies in area CA1. Sixteen to 20 hours after transfection, we imaged mCherry-expressing astrocytes for 15-20 min and established the rate of growth and volume changes of astrocytes. We then added either a glutamate agonist or antagonist for 30–40 min to determine how modulation of glutamatergic transmission affects astrocyte motility. Images were then reconstructed using 3D reconstruction software (Imaris, Bitplane) and rates of growth were analyzed.
Results: We observed highly-motile peripheral astrocytic processes which are partly actin-dependent (cytochalasin-D, 5 μM). Under conditions of increased activity (AMPA, 0.5 μM), we observed processes that retracted 4-10 μm at the periphery of astrocytes at a rate of approximately 1 μm/4 min compared to a rate of 0.6 μm/4 min prior to treatment. By eliminating evoked transmission (TTX, 1 μM), we observed an slight increase in motility of an approximate rate of 0.5 μm/4 min, while prior to treatment a generalized motility of 0.4 μm/4 min. Blocking AMPA-mediated transmission with NBQX (20 μM) did not have a significant effect on rate of growth (0.36 μm/4 min after treatment, 0.30 μm/4 min before treatment). Interestingly, addition of AMPA promoted astrocytic process retraction with directionality towards their central body. In addition, these retracting processes were polarized to one side of the astrocyte.
Conclusion: These results show that indeed mature astrocytes are sensitive to and respond to variations in synaptic activity. We are currently in the process of simultaneously visualizing astrocytic processes and dendritic spines to study how astrocytes induce activity-dependent structural modifications of mature synapses.
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