Category Archives: Optogenetics

ASCB15 – part 2

I ended Part 1 after the morning session on pushing the boundaries of imaging.

After the amazing talks on imaging, I browsed the halls, visited some exhibitors, sampled a couple of exhibitor tech-talks. I later went to a mycrosymposium (#2: signaling in health & disease). This was mainly to see how this ePoster thing works, but also I promised Qunxiang Ong – with whom I discussed optogenetics the day before – to be at his presentation. He used a light-induced dimerization of signaling proteins to study the effect on neurite growth. The nice thing in his system was that the cells were plated in wells which were partly dark – so light-induction cannot take place in these regions. This allowed for analysis of neurite growth in lit vs “light-protected” regions of the same cell.

After this session, I attended my first “discussion table”. Continue reading

This month’s Nature methods (part 2): optogenetics

Optogenetic tools are light-sensitive genetically encoded proteins that, upon light activation, affect a molecular change in the cells. In the previous post I described an optogenetic system to induce transcription. However, the most common use is of channelrhodopsin (ChR) molecules, that alter ion homeostasis upon illumination, and when expressed in neurons, can affect neuronal activity and – in live animals – a change in behavior.

Though optogenetics were used extensively in neurons in culture or in mice, its use was limited in flies. The reason was that the most common ChRs are activated by blue light, which does not penetrate well into the brains of live flies. Therefore, theremogenetic control has been used. In thermogenetics, a thermosensitive channel is expressed in the neurons. However, theremogenetics is far less accurate than optogenetics in terms of temporal resolution and intensity. On top of that, one needs to consider the effect of changes in heat on behavior.

To overcome that, the group of David Anderson decided to use a newly developed red-shifter ChR, ReaChr.

Using ReaChr, but not other Chrs, the researchers were able to activate specific neurons and alter fly behavior. Here’s one of their videos. Using this tool, they learned about the sexual behavior of the male fly and came to the conclusion that there are two sets of neurons which can be separated, with social experience affecting only one set of neurons.

The second optogenetic paper in this issue is more technical. The group of Edward Boyden decided to screen for new candidate ChRs in over 100 species of alga. They sequenced transcriptomes of 127 alga species and isolated 61 candidate ChRs which they tested for light-induced currents using electrophysiology methods. They chose 20 candidates and tested them in a range of wavelength excitations looking at current maxima, kinetics and more parameters.

characteristics of selected channelrhodopsins from different alga species. A, B, C, D - currents amplitude at different excitation wavelengths in HEK293 (A-C) and neurons (D).  E - Chrimson is the most red-shifted ChR. F - off kinetics. G - on-kinetics. H - recovery kinetics.

characteristics of selected channelrhodopsins from different alga species. A, B, C, D – currents amplitude at different excitation wavelengths in HEK293 (A-C) and neurons (D). E – Chrimson is the most red-shifted ChR. F – off kinetics. G – on-kinetics. H – recovery kinetics. Source: Klapoetke et. al. (2014) Nat. Meth. 11:338-346.

Of these, they selected two unique ChR: Chronos (with very high activity after blue or green light excitation) and Chrimson (the most red-shifted ChR – 45nm more red-shifted than ReaChR). After further characterization they showed that Chrimson is a good optogenetic tool for live flies and showed that they can stimulate neurons in fly brains.

Last, they created a two-color system. One of the limitations of the current ChRs is that all of them can be stimulated to some extent by blue light. Although Chrimson can be activate by blue light, the kinetics of Chronos are 10 fold faster. They did a very detailed work in finding the best conditions for a two-color system, based on the excitation pulse and power as well as expression level of the different ChRs.

All in all, a very nice work.

 

 
ResearchBlogging.orgKlapoetke NC, Murata Y, Kim SS, Pulver SR, Birdsey-Benson A, Cho YK, Morimoto TK, Chuong AS, Carpenter EJ, Tian Z, Wang J, Xie Y, Yan Z, Zhang Y, Chow BY, Surek B, Melkonian M, Jayaraman V, Constantine-Paton M, Wong GK, & Boyden ES (2014). Independent optical excitation of distinct neural populations. Nature methods, 11 (3), 338-46 PMID: 24509633
Inagaki HK, Jung Y, Hoopfer ED, Wong AM, Mishra N, Lin JY, Tsien RY, & Anderson DJ (2014). Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship. Nature methods, 11 (3), 325-32 PMID: 24363022
 

 

This month’s Nature methods (part 1): Spinach, blue transcription & photoacoustic imaging

This month’s Nature Methods issue has several interesting imaging items & articles, including two super-resolution reviews, two optogenetics articles, and more.

This post will be dedicated to three items in the “tools in brief” section.

Blue transcription

Optogenetics usually refers to control of ion flux via light sensitive channels. However, there are other light-responsive molecules. The item titles “Optimized optogenetic gene expression” describe a work from Kevin Gardner’s lab. They fused the transcription activating domain of the protein VP16 to the protein EL222. EL222 is a light-oxygen-voltage protein from the bacterium Erythrobacter litoralis. This protein binds to DNA when illuminated by blue light, and detaches from the DNA when the light is removed. Using this system, they could induce and repress transcription of a specific gene of interest (harboring a specific promoter recognized by EL222) in mammalian cells in tissue culture and in zebra-fish embryo. This can be a great tool.

Zebra fish egg and embryo harboring  an mCherry gene under the control of the VP-EL222 (or not) under dark or blue-light conditions. Source: Motta-Mena LB et al. (2014) Nat Chem. Biol. 10:196.

Zebra fish egg and embryo harboring an mCherry gene under the control of the VP-EL222 (or not) under dark or blue-light conditions. Source: Motta-Mena LB et al. (2014) Nat Chem. Biol. 10:196.

Photoacoustic imaging

Fluorescent molecules absorb light, and then emit light at a different wavelength. Photoacoustic molecules absorb light and emit sound waves. This is called the photoacoustic effect. This effect can be utilized to image inside whole animals, and the hope of the field is to get deep tissue penetration and a high resolution. The item titled “Activatable photoacoustic probes” presents a paper by the J. Roa’s lab at Stanford university. They developed a new polymer which absorbs at near-infrared (thus allowing good tissue penetration) and these produce a higher signal than commonly used materials for such imaging. They were also able for the first time to create a photoacoustic sensor of reactive oxygen species. This new field is very interesting and very exciting.

Spinach2

Spinach may deserve its own post, but briefly, Spinach and Spinach2 are RNA aptamers that can be used for the genetic encoding of fluorescent RNA. This aptamers form a unique structure which binds a specific molecule which then fluoresce. However, the optical properties of this dye were not suitable for common microscope filters. So now the group that developed Spinach developed several new dyes to enhance the fluorescent range of Spinach2.

The main problem I have with Spinach is that most of their work is based on an artificial RNA composed of 60 repeats of CGG trinucleotide and the ribosomal 5S rRNA. I haven’t followed the literature of Spinach much, but haven’t seen any single molecule imaging using Spinach. but, I guess I owe Spinach a post of its own.

ResearchBlogging.orgTools in brief (2014). Chemical biology: Optimized optogenetic gene expression Nature Methods, 11 (3), 230-230 DOI: 10.1038/nmeth.2867
Tools in brief (2014). Sensors and probes: Expanding Spinach2’s spectral properties Nature Methods, 11 (3), 230-230 DOI: 10.1038/nmeth.2865
Tools in brief (2014). Imaging: Activatable photoacoustic probes Nature Methods, 11 (3), 230-230 DOI: 10.1038/nmeth.2868
Pu K, Shuhendler AJ, Jokerst JV, Mei J, Gambhir SS, Bao Z, & Rao J (2014). Semiconducting polymer nanoparticles as photoacoustic molecular imaging probes in living mice. Nature nanotechnology PMID: 24463363
Motta-Mena LB, Reade A, Mallory MJ, Glantz S, Weiner OD, Lynch KW, & Gardner KH (2014). An optogenetic gene expression system with rapid activation and deactivation kinetics. Nature chemical biology, 10 (3), 196-202 PMID: 24413462
Song W, Strack RL, Svensen N, & Jaffrey SR (2014). Plug-and-Play Fluorophores Extend the Spectral Properties of Spinach. Journal of the American Chemical Society, 136 (4), 1198-201 PMID: 24393009
Strack RL, Disney MD, & Jaffrey SR (2013). A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. Nature methods, 10 (12), 1219-24 PMID: 24162923

Optogenetics blog

 Open optogenetics blog, is a blog about optogenetics. Optogenetics is a rather new field in neurobiology and fluorescent microscopy. The main idea of optogenetics is to have genetically encoded optically controled proteins (mostly, ion channels) and fluorescent sensors. I briefly discussed it here.
The blog discusses new sensors and other optogenetic research and papers. Recommended!

Sensing pH in neurons

A recent paper in Nature Neuroscience demonstrates the usefulness of the pH sensitivity of fluorescent proteins.

I have briefly mentioned the importance of pH when I discussed mKeima. Here I will describe the work from Richard Tsien’s lab which utilizes the effect of pH on FP in order to study synaptic activity.

One avenue of communication between synapse is vesicle exocytosis, i.e. the release of neurotransmitter-containing vesicles from the pre-synaptic cell. A surge of calcium ions in the postsynaptic cell follows vesicle exocytosis. Interestingly, when a vesicle is exocytosed, the lumen of the vesicle changes its pH from 5.5 to 7.4.

A pH sensitive GFP-based protein called pHluorin was developed as early as 1998. pHluorin was fused to a vesicle protein called VAMP2. Thus, a pH change from 5.5 to 7.5 increase the emission intensity (i.e.the protein becomes brighter when excited).

Although this is a good tool, the fact that it is GFP based limits the use of dual-color microscopy, particularly with other types of fluorescent sensors since most of them are GFP, CFP or YFP based, which makes spectral distinction difficult. Therefore, the authors set out to develop a RFP based pH sensor. After several rounds of mutagenesis and shuffling of mRFP and mStrawberry, they identified a bright pH sensor which they named pHTomato since its ex/em peaks (550nm/580nm) are similar to those of dTomato. Importantly, increasing the pH has increase the brightness of the protein, without affecting the ex/em peaks (unlike mKeima which I mentioned above). Furthermore, fusion to VAMP2 did not change its characteristics; the pH dependence is reversible (i.e. once pH is acidic again, the intensity decreases).

Since VAMP2 gives them a high background expression on the membrane, the authors decided to fuse pHTomato to a protein called synaptophysin (sy), which is more vesicle specific. sypHTomato was then compared to sypHluorin in the same neuronal cell. The authors show (by graph) that both proteins perform similarly. However, it would be nice to also show an image.

Once the authors establish that pHTomato is a good pH-sensor, they turn to the real exciting work of dual color sensing. As mentioned above, following exocytosis, there is a Ca2+ increase at the post-synapse. Electrical stimuli also cause a Ca2+ surge in the presynaptic cell. The authors then used a Ca2+ sensor called GCaMP3. Briefly, GCaMP3 is a GFP-based protein that was modified such that that the two part of the protein are fused to Calmodulin (CaM) and a peptide called M13. In the presence of Ca2+, CaM binds M13, thus bringing the two parts of the GFP together, which results in fluorescence (ex/em 489/509 – similar to EGFP).

Expressing both sensors in the same neurons allowed them to visualize the spike in Ca2+ (green spike) concurrent with an increase in red signal, that deteriorates slowly, upon stimuli to the neurons. I am not a neuroscientist, so I cannot evaluate their system regards to choice of cells/stimuli, but the fluorescence response following the different stimuli they give seems distinct and impressive. However, it is very difficult to see it in the snapshot images of the cells.

Expressing each sensor in a different cell allowed the authors to distinctly visualized pre and post synaptic cells at the same synapse. What they looked at are pre synaptic sypHTomato-positives cells, in contact with post-synaptic GCaMP3 positive cells. I think that the image is beautiful:

Figure 3: Dual-color imaging of synaptic connection by sypHTomato and GCaMP3. (partial figure)

Now that the technical issues have been address, it was time to ask some meaningful biological questions. The first question which they asked is whether the vesicle content at presynaptic termini (called boutons) of the same cell is the same in all synapses of the same cell, or are there differences based on the target, postsynaptic cell (obviously a pre-synaptic cell can create synapses with multiple post-synaptic cells).

They used stimuli to measure the volume of the readily-releasable vesicles, and ammonium chloride (NH4Cl), a strong base, to measure the total volume of all vesicles in each synapse. They found that synapses that were targeted to the same postsynaptic neuron had reduced variability in total and readily-releasable vesicle volume, compared to the variability of synapses targeted to different cells. This indicates that there is some reverse feedback from the post-synaptic cell to the pre-synapses.

It would have been interesting to see if pre-synaptic boutons from different cells also have less variability if they are targeted to the same post-synaptic cell. However, the authors did not measure that.

They then looked what happens when you stimulate the cells electrically. As ecpected, there is an immediate increase in sypHTomato signal (vesicle exocytosis) with an almost immediate increase in GCaMP3 signal (Ca2+surge) in the post synaptic cell. This Ca2+ surge begins at the synapse but propagates along the dendrites, indicating opening of voltage-gated Ca2+ channels with the propagation of the ation potential. They confirmed that the Ca2+ surge is due to the vesicle release by adding inhibitors for the neurotransmitter receptors on the post-synaptic cell, and detecting little or no Ca2+ increase (The inhibitors did not affect vesicle release). This approach, then, was capable to image neuronal activity at single synapse resolution.

But the authors went one step further, to get achieve an all-optical system.

The experiment described above was performed by electrically stimulating the cells. However, such methods are invasive and affect the entire cell. An alternative strategy to activate cells is by chemical application of agonists or antagonists. However, addition of drugs or neurotransmitters to the cell culture media will affect all cells in the media. Here comes optogenetics – a system that allows optical activation of a single cell, or part of it. I do not want to discuss optogenetics here; this should get a post or two of its own. The basic idea is to use genetically encoded light-responsive ion channels. Once you shine light (at a specific wavelength) on the part of the cell you want to activate, the ion channel opens and you get action potential originating from the part of the cell you shined on. So here comes the cool stuff:

The authors co-expressed channelrhodopsin2 (ChR2) with a pHluorin-tagged vesicular glutamate transporter (vGluTpH). ChR2 gets to the plasma membrane, whereas vGluTpH to the vesicles. When they shined blue light (to activate ChR2 and excite pHluorin) they get an increase in the green signal, which is abolished by a drug that inhibits action potential. Similarly, co-expression of VChR1 (another optogenetic tool) with stpHTomato and shining green light (to activate VChR1 and excite pHTomato) led to an increase in red emission, that was abolished by the drug.

Better yet, co-expressing ChR2 with sypHTomato showed an increase in red signal only when cells were illuminated by blue light (that activates ChR2) and not green light (Which doesn’t). This figure should have been a main figure. I don’t know why they put it in the supplementary.

Fig S6(b) Tests of ChR2 in combination with sypHTomato as proof-of-principle of all-optical yet independent monitoring and stimulation. Left, interrogation of sypHTomato with Green light (546 -566nm) excitation alone without activation of ChR2-driven vesicular turnover. Right, positive control with blue light (457-482 nm), showing robust ChR2-driven sypHTomato transient (right).

And there you have it – an all optical system to study vesicle release in neurons.

So what can we do with this system?

The authors state several exciting possibilities:

  1. Perform dual color experiments with other green sensors.
  2. Deciphering pre and post synaptic strength in different scenarios.
  3. Probing neuronal circuits: combining optogenetic photostimulation of two spectrally distinct channlerhodopsins with two spectrally distinct sensors (pH sensors in this case) will allows us to follow the neural pathway when we activate a distinct neuron with a specific color.

But I could think of other options. For instance, the authors did not discuss at all the growing use of caged molecules. In brief, caged molecules are biologically relevant molecules that are in an inactive state. These molecules can be activated by shining light of a specific waveband. Thus, if you have a caged neurotransmitter and you shine the light at a specific location, synapses can be activated only if the uncaged molecule is at high enough concentration (i.e. where you shined your light).

The authors kind of ignored it, but there seems to be a difference in the sypHTomato intensity, and signal decay, when comparing electrical and optical stimulation. This difference could be biologically meaningful and could be further explored.

All in all, I think it is a very nice paper, describing a new system to study neurons.

Further reading:

Yulong Li & Richard W Tsien (2012) pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity. Nature Neuroscience 15, 1047–1053.

A good post on GCaMP at “brain Windows” blog.

Optogenetics resource center

Info on chemical synapses, from Wikipedia

ResearchBlogging.org
Li Y, & Tsien RW (2012). pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity. Nature neuroscience, 15 (7), 1047-53 PMID: 22634730