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





The great GECI shootout

21 07 2008

Dierk Reiff’s lab has done another head-to-head in vivo showdown between various GECIs and a synthetic dye. Their paper, Fluorescence changes of genetic calcium indicators and OGB-1 correlated with neural activity and calcium in vivo and in vitro, is very interesting and deserves a full write-up. I will present a detailed analysis of the paper in a future update.  For now, check the abstract.

Recent advance in the design of genetically encoded calcium indicators (GECIs) has further increased their potential fordirect measurements of activity in intact neural circuits. However, a quantitative analysis of their fluorescence changes ({Delta}Fin vivo and the relationship to the underlying neural activity and changes in intracellular calcium concentration ({Delta}[Ca2+]i) has not been given. We used two-photon microscopy, microinjection of synthetic Ca2+ dyes and in vivocalibration of Oregon-Green-BAPTA-1 (OGB-1) to estimate [Ca2+]i at rest and {Delta}[Ca2+]i at different action potential frequencies in presynaptic motoneuron boutons of transgenic Drosophila larvae. We calibrated {Delta}F of eight different GECIs in vivo to neural activity, {Delta}[Ca2+]i, and {Delta}F of purified GECI protein at similar {Delta}[Ca2+in vitro. Yellow Cameleon 3.60 (YC3.60), YC2.60, D3cpv, and TN-XL exhibited twofold higher maximum {Delta}F compared with YC3.3 and TN-L15 in vivo. Maximum {Delta}F of GCaMP2 and GCaMP1.6 were almost identical. Small {Delta}[Ca2+]i were reported best by YC3.60, D3cpv, and YC2.60. The kinetics of {Delta}[Ca2+]i was massively distorted by all GECIs, with YC2.60 showing the slowest kinetics, whereas TN-XL exhibited the fastest decay. Single spikes were only reported by OGB-1; all GECIswere blind for {Delta}[Ca2+]i associated with single action potentials. YC3.60 and D3cpv tentatively reported spike doublets. In vivo, the KD(dissociation constant) of all GECIs was shifted toward lower values, the Hill coefficient was changed, and the maximum {Delta}F was reduced. The latter could be attributed to resting [Ca2+]i and the optical filters of the equipment. These results suggest increased sensitivity of new GECIs but still slow on rates for calcium binding.





Sensing salty currents with Mermaids

16 07 2008

A new genetically-encoded voltage sensor paper is out from a friend and former mentor of mine, Atsushi Miyawaki. One memorable moment when working in his lab during the RIKEN summer program of 2002 was when Atsushi took me into his office and whipped out a custom green laser pointer. These had been banned in Japan, as fans would shine their powerful light into the eyes of pitchers and batters at baseball games. Atsushi was really proud of his. He smiled and then started sweeping the light point over the rocks in his fishtank. Each ‘rock’ was actually coral his lab had collected from fluorescent protein hunting trips, and each glowed a different color when the green light hit it. He has been putting these novel discoveries to good use.

In Improving membrane voltage measurements using FRET with new fluorescent proteins, Tsutsui et. al take two fluorescent proteins discovered and engineered by the Miyawaki lab, mUKG and mKOk, and graft them onto the Ci-VSP scaffold used in VSFP2.1 (also developed at RIKEN).  The green and orange fluorescent proteins undergo significant FRET transfer which is voltage dependent.  They get 40% dR/R per 100mV with a 2 component association rate of around 10 and 200ms. Unsurprisingly, the kinetics speed up at physiological temperatures to 5-20ms on and off.  They are able to pick up single pseudo-action potentials in Neuro2A cells, though the response is highly filtered. They are also able to see very clear spontaneous waves of potential change in cardiomyocytes (23% dR/R) and single spikes in cultured neurons (2% dR/R for 1AP). They dub this voltage sensor “Mermaid”.

The authors state that they used the new FPs due to their improved photostability and especially pH resistance. 

Additionally, because Aequorea GFP variants are pH-sensitive, and neuronal activity causes considerable acidification, the responses of sensors to depolarization in intact neurons may be overwhelmed by sustained changes resulting from acidification.

Granted that mOrange2 is pretty pH-sensitive, but I’m not sure this is a real issue, or a potential issue to justify using their new FPs.  From the spectra of mUKG vs. EGFP, it would seem that EGFP’s 10nm further redshifted emission would be a superior FRET pair for mKOk.  It smells like there may be a bit of bundling of various independent projects into this paper.  However, they do make a good point that this pair will have a different preferred dipole orientation than existing FRET pairs, which could lead to improved performance in some constructs.  

Things I’m still wondering :

  • Have they tried using the improved VSFP3.1 scaffold? This was shown to be much faster than 2.1.  I suspect the mUKG is not as tolerant to C-terminal truncation than CFP and GFP.  
  • What about using EGFP as the donor?  Could you then use the VSFP3.1 scaffold?
  • Is there a rapid non-FRET quenching of the donor upon depolarization as seen in VSFP3.1?
  • Why is the single wavelength fluorescence increasing in both channels in figure 2d?  Is there some photoactivation going on?
  • I’d love to see a head to head comparison of VSFP3.1 and Mermaid under identical conditions. Also responses in brain slice at physiological temperatures.




Voltage sensitive imaging powering up

8 07 2008

I’m starting to come around on voltage imaging. I haven’t been a fan of it for a number of reasons.

  • The response sizes suck.  Classic dyes and genetically encoded systems get a few percent fluorescence change at best. 
  • The response speeds suck. Measuring continuous current injections from -100mV to +150mV is not very interesting.  Action potentials are interesting.  But they are fast.
  • Toxicity. The dyes kill neurons, or strongly perturb their electrical properties.

OK, voltage-sensitive imaging isn’t totally useless, for example see Carl Petersen’s recent paper on Spatiotemporal Dynamics of Cortical Sensorimotor Integration in Behaving Mice (2007). But if the above problems could be solved, then voltage sensitive imaging would be a strong competitor to calcium imaging for the non-invasive, high-resolution monitoring of patterns of network activity. There has been considerable progress ameliorating these problems in the past few years, much of it by a consortium of labs (Isacoff, Knöpfel, Bezanilla, Miesenböck, and others) focused on these issues. (Umlaut’s apparently help in this field).

First, let’s look at a minor breakthrough for the fully genetically-encoded strategy.  In Engineering and Characterization of an Enhanced Fluorescent Protein Voltag Sensor (2007), The Knöpfel group tagged the recently discovered voltage sensitive phosphotase (Ci-VSP) with CFP and YFP FRET pairs in place of the phosphotase domain. This tagged protein expressed at the membrane much more efficiently than previous genetically encoded voltage sensors based on potassium channel subunits. By injecting physiological voltage changes and averaging 50-90 traces, they were able to pull out a few percent ratio change from a brief series of action potentials. Single spikes were resolvable.  Although this sensor (VSFP2.1) was pretty slow (tau > 10ms), this new substrate looked promising for future sensor development.

They have since sped the response up.  In Engineering of a Genetically Encodable Fluorescent Voltage Sensor Exploiting Fast Ci-VSP Voltage-Sensing Movements (2008 ), they determined that the gating motion of the voltage sensing component was very fast (~1ms), while the fluorescence change was slow (~100ms). So they did what any good FRET tinkerer would do, chop away at the linkers between FP components.  The sensor response improved, and they noticed that there was a disconnect between the speed of the CFP and YFP responses. Not only was CFP decreasing from enhanced FRET, it was being directly quenched by interactions with the lipid membrane. Chopping off the YFP from the the construct then dramatically increased the speed of the CFP quench. This improved sensor, VSFP3.1 has an activation time constant of 1.3ms, though it’s response magnitude is still quite small (a few % dF/F).

A hybrid approach to measuring electrical activity in genetically specified neurons (2005) has a much greater response magnitude. Pancho Bezanilla’s group exploited the rapid, voltage-dependent translocation of the small molecule quencher dipicrylamine (DPA) through the plasma membrane to change the fluorescence of membrane-teathered GFP in a voltage-dependent manner. Responses of the hybrid voltage sensor (hVOS) were relatively large (34% per 100mV) and fast (0.5ms). Single action potentials were detectable without averaging.  However, since DPA is a charged molecule, it significantly increased the capacitance of the membrane. The levels of DPA required to see large responses inhibited action potentials and were intolerable to neurons.

Last month in Rational Optimization and Imaging In Vivo of a Genetically Encoded Optical Voltage Reporter (2008 ), Sjulson and Miesenböck reported optimized parameters for the hVOS approach. They built a quantitative model of the quenching effects of DPA on membrane-teathered GFP.  The quenching is limited by the distance the DPA can approach the chromophore of GFP.  Only the closest DPA molecule to the chromophore significantly contributes to a GFP’s quenching. After lots of pretty heat maps and graphs, the model tells them to chop off the tail of EGFP to bring the C-terminal tethering sequence closer to chromophore. I should note that an 11 amino acid C-terminal truncation of ECFP has improved the response of a tremendous number of FRET reporters and has been standard practice for the last 8 years. By shortening the linker they manage to triple the response size. I’d suggest, if they haven’t already, to lop off another six amino acids (end the EGFP with …LEFVTAA) and see if works.  EGFP and ECFP usually tolerate it.

Using this optimized reporter, they are able to reduce DPA concentrations to levels that are usable in vivo, at least for a few minutes. They record fast optical responses to electrical activity in the Drosophila antennal lobe using 2uM DPA.  But after a few minutes, the DPA loaded neurons become strongly inhibited.

 

The bottom line? Voltage-sensitive imaging has seen big progress in the last few years, but still has a long way to go to gently record single APs in a dish or in vivo. Or does it?  I’m hearing whispers that a different group has developed a synthetic dye technique that is getting >10% dF/F to single APs with millisecond response times. Is it the real deal? Watch this space…





SLICK labeling and new FPs

1 07 2008

There is a nice writeup of the single-neuron labeling with inducible Cre-mediated knockout (SLICK) paper from Guoping Feng‘s lab over at the Alzheimer’s Research forum. The method simultaneously knocks out a gene in a small number of cells, while highlighting the knocked-out cells with a cytosolic fluorescent protein. In a comment to the Schizophrenia Research Forum, Joseph Gogos points out a similar technique his lab published last year in Current Biology.

Also in the writeup is coverage of the new fluorescent protein variants from the Tsien Lab.  These include mOrange2 made by Nathan Shaner, which is a much more photostable version of mOrange. This should immediately replace mOrange in most constructs.  Also of note is TagRFP-T from Michael Lin and his trusty undergraduate assistant Michael McKeown. Tag-T is an extremely photostable derivative of the Evrogen protein TagRFP. Tag-T was discovered by screening Tag mutants in bacterial colonies on a solar simulator. Toxicity in sensitive cells (in vivo neurons) hasn’t been fully determined yet, but in vitro these new FPs all look great. Now I wish they would make a super-bleach resistant Citrine for my FRET constructs.





Giving synapses a ‘born on’ label

30 06 2008

Memories are thought to be encoded by the patterns of synaptic connections in the brain. Learning can either delete or change the strength of existing synapses, or add new synapses. Following a learning process, how can we tell which synapses were added to encode this new memory?  

One strategy is to make a timelapse movie of the synapses.  In mice, this can be accomplished by installing a cortical window on the skull, and imaging the changes in structure of GFP labelled neurons. However, this is technically demanding, only works with sparsely labeled neurons, and accesses only a small subset of the neurons which may be involved in the learning process.  

Ideally, one could have a tag which can discriminate between synapses existing before learning takes place, and new ones generated after learning has occurred. Whole brain regions could then be examined at a single timepoint to see where new synapses were added. In a large step towards that goal, Michael Lin et. al, from the lab of Roger Tsien, report TimeSTAMP, a genetic label for newly synthesized protein.

The authors engineered the NS3 protease from the hepatitis C virus (HCV) to cleave itself at just the right pace. They then fuse tags (fluorescent proteins or epitopes) before and after the cleavage site. This fusion is then tagged to the end of a protein of interest. Shortly after synthesis, the protein cleaves off the C-terminal tag, but the N-terminal is left on. This cleavage is inhibitable by a variety of small molecule blockers. In the presence of the blocker, the C-terminal tag stays on. By controlling when drug is applied, they can selectively label a set of proteins of a particular age with the tags.

The choice of NS3 protease was very clever, as it is a favorite drug target of biotech and pharma companies.  Many inhibitors of this protein have been synthesized, exhaustively characterized in vitro and in clinical trials. This work is a great example of the standard research flow going in reverse; a basic-science project from an academic lab is actually benefitting from pharma company research. Stability, bioavailablity and toxicity have already been worked out.  One of the biggest impediments is actually getting ahold of these compounds. Companies with their survival hanging on the clinical success of a single small molecule inhibitor are understandably reluctant to hand out stocks for academic research. Note the roller coaster stock price of Vertex following results of its NS3 protease inhibitor (VX-950) trials. 

The authors use PSD-95 tagged to TimeSTAMP as a proxy marker of synaptic age. In neuronal culture, they show that newly synthesized synapses have a C-tag / N-tag ratio of about twice as large as old synapses.

They extend the technique to whole fruit fly brains, showing a very heterogeneous distribution of CaMKII synthesis across Kenyon cells in different areas of the mushroom body.

So far TimeSTAMP has not been shown to work in mice. Mice were not included in the paper due to the long generation time for transgenics. Given the good signal to noise and the large number of possible inhibitor molecules, I think this technique could be quite powerful in mammalian systems. It’s big advantage would be to label large populations of neurons or synapses in diverse brain regions, including those inaccessible to two-photon microscopy. TimeSTAMP’s success in labeling new synapses in the intact brain will be dependent on finding a protein to tag at the synapse with low turnover over the course of a learning experiment. Though PSD-95 appears to be a reasonable marker in culture, others have shown a higher rate of turnover in vivo, making in unsuitable for a synaptic marker. 








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