When I was working on my Ph.D. thesis, I was trying to find some biological question to definitively answer with GluSnFR, my glutamate sensitive fluorescent reporter. One possibility was the study of glutamate release from astrocytes. Around that time, 2003/2004, there was increasing evidence that glutamate was not just scavenged by astrocytes, but was also released from astrocytic vesicles. It released in response to calcium elevations within the cell. Existing methods for measuring this release were somewhat crude, so it seemed a great test system for GluSnFR.
Unfortunately, since there seemed to be no specialized areas on the astrocyte where the vesicles fused, and the release rate was relatively slow, we were unable to detect glutamate release with GluSnFR. I thought this might be a problem of not knowing when and where to look. So my collaborator, Yongling Zhu, and I expressed pHluorins fused to VAMP or to synaptophysin in astrocyte cultures. When we looked at them under the microscope, they just looked green, no action…
But then we left the excitation light on for a few minutes. I happened to look back into the scope after they had been bathing in bright blue light and was astonished. I could directly see, by eye, spontaneous bursts of fluorescence across the cells. It was absolutely magnificent. The long application of light had bleached all of the surface expressed, bright pHluorins. But the pH-quenched pHluorins in the vesicles were resistant to bleaching. On this dimmer background, the fusion events were plain as day.
Unfortunately, the green color overlapped with the emission of GluSnFR, so we couldn’t use it for a spatiotemporal marker of when and where to look for glutamate release. We tried using some ph-sensitive precursors to mOrange and mOrange2, developed by Nathan Shaner, but these seemed to block the release events. Since then, others have shown the functional relevance of glutamate release from astrocytes, and I turned the focus of GluSnFR measurements to synaptic spillover. This was one of the projects that was tantilizingly close, but got away. This movie of VAMP-pHluorin is almost five years old now, but it still looks cool… Enjoy!
If you are curious, this is what the Synaptophysin-mOrange looked like when we expressed it in hippocampal neuron cultures. Ammonium Chloride caused a massive fluorescence increase, by alkalizing the synaptic vesicles. Unfortunately, we never were able to see release via electrical stimulation. Details are in my thesis. Maybe someone else wants to give it a shot?