The Effects Of Astrocytes On Neuron Dendritic Filopodia Motility

Background and Objective:It has been reported that astrocytes could modulate neuron generation, spine formation and message transmission by releasing some gliatransmitters such as glutamate, ATP and D-serine. These gliatransmitters have inhibitory effect to synaptic junction. The motility and morphology of dendrite spine relate with synaptic junction, but the detail for astrocytes effects on dendrite spine is still not clear. Recently, some labs found that astrocytic protrusive activity acts as a key local regulator for stabilization of individual dendritic protrusions and subsequent maturation into spines. However it is still a problem whether astrocytes could regulate filopodia motility and development by releasing gliatransmitters. Here we use a special tool Channelrohdopsin-2, with which we could activate astrocytes selectively in neurons and astrocytes mix-cultured model. We try to explore how does astrocytic activity effect drendrite filopodia motility.Methods:Cortical astrocytes and neurons were prepared from SD rat, and a mix-culture model of astrocytes and neurons was established. Astrocytes were transfected with ChR2 cDNA by liposome. We stimulated astrocytes with a blue light by a special model (1Hz,800ms open,200ms closed, and 10 pulses every time). At the same time we took a timelapses picture of astrocytes calcium wave change by confocal laser scanning microscope. Pure neurons were transfected with GFP by electrotransfection method. On the fourth day we imaged morphology change of neuron dendritic filopodia. We took three groups in the experiment, control group, group administrated with 100μMATP and group administrated with 100μM glutamate. The last section of our experiment is activating astrocytes by blue light in the mix-cultured model and imaging neuron dendritic filopodia motility. We made a quantitative analysis of the filopodia motility by metamorph software and checked the effect of ATP, glutamate and astrocytic activity on filopodia motility.Results:1 Channelrhodopsin-2 could be expressed on astrocytes membrane for one week. We made a special stimulating model (1Hz,800ms open,200ms closed, and 10 pulses every time), which could activated astrocytes at a higher rate than other protocals. Astrocytes showed some different patterns of calcium wave under the stimulating model. The calcium waves change patterns include a single pulse of rapid calcium wave, multiple pulses of rapid calcium wave, one pulse of slow calcium wave and on-off pulses of calcium wave.2 100μM ATP or 100μM glutamate separately inhibit filopodia motility.3 In the mix-cultured model, activated astrocytes also inhibit neuron dendritic filopodia motility.Conclusion:Astroytes with ChR2 was activated by a special blue light. Astrocytes showed some different patterns of calcium wave under the stimulating model. Activated astrocytes inhibited neuron dendritic filopodia motility as well as by administrating with ATP or glutamate. The results demonstrate that inhibitory effect of activated astrocytes on filopodia motility maybe induced by ATP or glutamate, which could be released by astrocytes in vivo.