Software Update : Ephus, ScanImage & Neuroptikon

20 08 2010

Three excellent pieces of neuroscience software have been recently updated or freshly released.  I have used two of them, Ephus and ScanImage, on a daily basis as primary data collection tools. The third, Neuroptikon, is quite useful for post-hoc illustration of neural circuits.

Ephus is a modular Matlab-based electrophysiology program that can control and record many channels of tools and data simultaneously.  Under control of a sophisticated internal looper or external trigger, you can initiate an ephys recording, trigger camera frames, adjust galvo positions, open/close shutters, trigger optical stimulation, punishments, rewards, etc.  It is a workhorse program for non-imaging related in vitro and in vivo electrophysiology experiments.  Ephus is named for the fabled baseball pitch, and pronounced as “EFF-ess”. As with the pitch, it may trick you at first, but eventually you’re sure to hit a home run. Of course, the name also evokes electrophysiology, which is the fundamental orientation of the project, be it optical or electrical.

Ephus 2.1.0 is a major release, and the only official version at this time.  The software is fully described in a publication in Frontiers in Neuroscience. New features include unlimited recording time, with disk streaming, for applications such as EEGs and long traces during in-vivo behavior. A number of additional scripts for in-the-loop control have been added. New configuration/start-up files have been created, with a template to help get up and running quickly. This release also includes a number of bug fixes.

ScanImage is another Matlab-related software program that is used for optical imaging and stimulation of neurons in vitro and in vivo.  It finds much use a control platform for 2-photon imaging, glutamate uncaging and laser-scanning photostimulation.  An early incarnation is described in this paper by Pologruto, et al.  It provides a lot of power right out of the box (bidirectional scanning @ 0.5ms/line, etc) and is easily extensible via custom user function plugins.

Neuroptikon is a sophisticated network visualization tool.  It can build Van Essen-like diagrams of any circuit you like, but it is so much more.  The direction of communication is animated, and subsets of regions and connections can be brought into focus, which greatly eases the clarity of the network.  The diagrams can be built in three-dimensions, to preserve relative topography, or functional grouping.  There is simple GUI-based control, while more complex tasks can use a scripting interface.  This is great software for anyone who needs to imagine information flow in a complex network.

All three tools are released for free use under the HHMI/Janelia Farm open source license.

Download Here :

Ephus 2.1.0

ScanImage 2.6.1

Neuroptikon 0.9.9

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Raw Data : Vesicular Release from Astrocytes, SynaptopHluorange

15 11 2008

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?





2008 Nobel Prize in Chemistry to GFP

8 10 2008

This morning, the Nobel committee recognized the work of Osamu Shimomura, Martin Chalfie and Roger Tsien “for the discovery and development of the green fluorescent protein, GFP” by awarding them the Nobel Prize in Chemistry for 2008.  A video of a great lecture on fluorescent proteins by Roger Tsien is available here.

Green Fluorescent Protein

Green Fluorescent Protein

Shimomura first discovered GFP during the study of the bioluminescent protein aequorin, the mechanism by which certain jellyfish glow.  In the footnote to his seminal paper on aequorin purification, he noted the additional presence of “a protein giving solutions that look slightly greenish in sunlight through only yellowish under tungsten lights, and exhibiting a very bright, greenish fluorescence in the ultraviolet of a Mineralite, has also been isolated from squeezates.” 

Aequorea Victoria

Aequorea Victoria

Chalfie took the cDNA of GFP and first expressed it bacteria and worms.  He demonstrated GFP could be used as a molecular tag. Surprisingly, the protein folded and functioned without the use of co-factors specific to the jellyfish.

Tsien developed GFP into the many useful variants we use today.  He reported the S65T point mutation that greatly improved its fluorescent characteristics. His lab also evolved GFP into many other color variants, and demonstrated that these variants could be used as genetically-encoded intracellular sensors for calcium, enzyme action, and glutamate.

Chromophore of the S65T mutant of GFP

The odd man out in this triumvirate is Douglas Prasher.  With a tiny lab and budget, Prasher discovered the primary sequence of GFP and cloned the cDNA of GFP. Unfortunately, around the time of his work’s publication, his grant ran out. Prasher sent out DNA samples to Chalfie, Tsien and others, shut down his lab and left science. Prasher’s contribution was the essential foundation for the explosion of developments in the field.

Some argue that Tsien would have already won the Nobel prize for calcium signaling if not for his contribution to GFP. As a graduate student, he invented the high affinity calcium chelator BAPTA. Using BAPTA as a foundation, he created a large family of fast, bright calcium dyes, including fura-2.  Nearly every fluorescent dye for calcium was either his invention or a close variant of one of these. The importance of these tools for understanding intracellular communication cannot be overstated.

Transgenic GFP mouse





Optical imaging of neuronal glutamate release and spillover with GluSnFR

12 03 2008

This post is difficult to craft. I’ve been struggling with whether to write an epic post describing the history of glutamate imaging, the major advances and players in the field and where I fit into it, or a simple post focused on my new paper. Since glutamate imaging is my field, I’ve got tons to say about it, but also there is probably no way to avoid significant personal bias in my account. So, I’ll go with the short form. For those interested in further reading, please check out these earlier reports, including our brief mention of neuronal glutamate measurements with GluSnFR prototypes, neuronal glutamate measurement with FLIPE and the optimization of FLIPE constructs from Wolf Frommer’s group, and the use of FLIPE’s in brain slice to look at broad patterns of glutamate release from the Huguenard group.

In this PNAS paper, Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters, by Hires et al. from the group of Roger Tsien, the authors report on the optimization of GluSnFR, a genetically-encoded Glutamate Sensitive Fluorescent Reporter, and its application to the study of glutamate spillover. A cyan and yellow fluorescent protein bracket a glutamate periplasmic binding protein. Glutamate binding to the PBP causes a conformational change and a reduction in the amount of FRET between the fluorescent proteins. Glutamate concentration can be quantitatively determined by observing the ratio of the blue to yellow fluorescence. When fused to an extra-cellular membrane targeting motif and expressed in neurons, optical responses to synaptic glutamate release were detected.

glu_response.jpg

FRET constructs are very fickle, with their response being very sensitive to how the sensor components are fused together. This paper clearly demonstrates this, as 176 linker combinations were screened for maximal ratio change and only one was far superior to all others. The optimized SuperGluSnFR showed a 44% ratio change between zero and saturating glutamate levels in Ringer’s solution, a 6.2-fold improvement over the original prototype. Importantly, the screen took place in a system, HEK cells with surface displayed GluSnFRs, that was physiologically similar to the neuronal system where the sensor was ultimately used. This ensured that the screen discovered useful improvements, rather than ones that worked great in the screening system, but did not express or respond well when expressed in neurons. Previous glutamate sensor optimization in bacteria lead to large responses in vitro that did not translate well when ported to surface-displayed plasmids. Note though that this optimized sensor, FLI81PE, has found use when bath applied to brain slice.

grid.jpg

SuperGluSnFR was used to address questions about glutamate spillover. Under what conditions might glutamate spill beyond the synaptic cleft? How long does this spillover last, and what effects might it have? The paper makes the first direct, quantitative measurements of the timecourse of glutamate spillover. It shows that, at least in cell culture, spillover following burst stimulation can cause a significant glutamate transient along the entire dendritic surface, not just at the synaptic active zone. After a single action potential, spillover is insufficient to activate any extrasynaptic glutamate receptors. But, after a burst of stimulation, sub-micromolar glutamate levels persist long enough to activate extrasynaptic NMDA receptors. This could have a tremendous impact on dendritic computation, synaptic independence and heterosynaptic long term potentiation or depression.

glucurves.jpg

There are two significant limitations to the conclusions of this paper. First is that the experiments were done in dissociated hippocampal culture at room temperature. Glutamate transporters are faster at physiological temperatures, and the geometry of the neuropil in vivo might reduce the impact of spillover. Secondly, there is no electrophysiology to directly support the NMDAR activation assertion. Hopefully, some other group will pick up this thread and do more rigorous testing. GluSnFR imaging in acute brain slice should be easy enough using in utero electroporation techniques, and many, many labs have the electrophysiology experience needed. I’m tempted to do the experiments myself, but there is simply no time!

If anyone would like to try SuperGluSnFR for their own work, send me an email and I’ll be happy to send out an aliquot!