- Arjun A Bhaskaran added an answer:8Does anyone have experience using WGA-CRE virus to activate DIO opsins in projecting regions?
Our lab is in the process of setting up in vivo optogenetics in RATS for behavioral studies. The goal is to activate specific projections based on their connectivity, so we are considering the method of injecting the opsin into region A and a WGA-CRE virus into region B, so that when we stimulate A, only those cells projecting to B get activated.
Is this method reliable enough to get good expression of the opsin? Are there issues with spillover? We are planning on using AAV5 for the opsin to get a large region of transfection. Is AAV5 the best serotype to use for WGA-CRE as well, or would something like AAV9 work better?
How does this method compare with using CRE packaged in a retrograde virus (e.g., CAV, PRV)?
We use RABV (glycoprotein free) for the retrograde and anterograde tracing. I do not know you can combine opsin with this virus and inject in the specific region!Following
- Shan Shen added an answer:4Does anyone have any experience with UHP-Mic-LED-595 - Ultra High Power LED Light Source from prizmatrix?
I was trying to find an high-power LED to drive the ArchT optogenetically. Two thorlabs LED (590nm and 595nm) have been too weak for us and we found a high power one on prizmatrix. The representative said that this model might cause some noise in Ephys recording. Does anybody have experience with that?
Thanks Fernando! That's exactly the info I need.
- Boaz Barak added an answer:6Is anyone using ANY-maze software for tracking the animal and triggering the laser in optogenetic behavioral experiments?
I'm planning to set up the optogenetic stymulation system in behaving animals and just now I'm considering some software options. I'm currently hesitating between ANY-maze and Ethovision.
Anybody is using any of these softwares?
I know someone who worked with Ethovision and claims the software does its job correctly but this one is extremely expensive and every other 'module' or upgrade costs few thousands $. ANY-maze seems to be doing similar job for around 7k$ with everything included, but I don't know anybody using it so I don't know if there are some problems that come up only later.
I would very much appreciate any comments concerning your experiences and/or difficulties encountered with both or any of the softwares.
I did something similar with Ethoviosion (Noldus), so it is not exactly what you asked, although the concept is similar.
What you want to do is connect your PC (with the Any maze software installed) with a cable to the input slot in a TTL box. Then you can connect from the output slot of your TTL with a cable to either pulse generator (like Master9 for example) and then from it to the laser head, or directly from the TTL to the laser head.
- Roni Hogri added an answer:13Are there published studies which used retrograde transfection to express opsins in upstream neurons?
If you have a heterogenous neuronal population in area A who are projecting to areas B and C, you might want to transfect area B with opsins and then have the opsins expressed retrogradely in the somas of cells in area A - then you could shine the light at area A and know that you excite (or inhibit) the firing of neurons projecting to area B (and not C), while avoiding problems related to axon terminal stimulation. I've read of retrograde genetic manipulations, but I've not found anything specific to optogenetics. Are there any papers out there showing the use of retrograde transfection in optogenetic studies?
Since it's 2 different sources and the expression itself (at the site of injection, and in axons at sites to which the transfected neurons project) is very nice for both of them, I doubt the problem is the source. The difference may be in the species, the use of Cre, and/or brain areas...Following
- Evelyn Ploetz added an answer:6A reliable laser supplier for in vivo optogenetics?
Hi all. We have been performing in vivo optogenetics for some time now, using DPSS lasers to provide our light source. We find the power of these lasers depreciates quite rapidly (a 100 mW laser can be putting out only 50 mW a few months after purchase), so I would like to know if anyone can recommend a manufacturer that provides good quality, stable lasers. Thanks!
we have good experience with the Coherent OBIS Laser family (375,405,488,532,640). We are using them since 4 years . Stable output power, can be use in CW and pulsed mode...Following
- Eoin C O' Connor added an answer:10Should I use AAV serotype 2 or 5 for expression of ChR2 in the ventral tegmental area?
Leaning toward serotype 5. I have read that serotype 5 is better in adult rodents. That was outlined here
Han X. Optogenetics in the nonhuman primate. Progress in brain research. 2012;196:215-233. doi:10.1016/B978-0-444-59426-6.00011-2.
Seeing if anyone has experience handling these vectors and injecting them.
Please don't forget that the promoter also makes a huge difference to expression levels. We have used AAV5-EF1A based constructs with a lot of success (including in mouse VTA).Following
- Leslie M. Kay added an answer:1Experience with Prizmatix LEDs for in vivo optogenetics?Their products look both interesting and capable for our purposes. Has anyone here first hand experience with Prizmatix products? If so, which ones?
Did you ever get an answer on this?
I am looking at their products now.Following
- Alessandro Bilella added an answer:15DREADD vs. optogeneticsI am thinking of employing both these techniques in my lab and was wondering if people would like to share their thoughts/experience.
From a behavioural point of view I suppose that a major advantage of optogenetics is that you have greater temporal control over stimulation: once a dose of CNO is administered to an animal expressing DREADD there is presumably a time-to-onset and later a decline in receptor occupation and effect, whereas with ChR2/NpHR simulation is phase-locked to light stimulation.
By contrast, I would imagine that light scattering (optic fibre in brain)/failure of light to penetrate tissue sufficiently (stimulation of peripheral nerves in skin) is a drawback of optogenetic stimulation compared to oral administration of CNO, which has known efficacy at different DREADDs.
Any thoughts/comments welcomed!
Yes, in both cases is Cre-recombinase.Following
- Fengling Li added an answer:10Any suggestions on the abnormal activities of V1 neurons in PV-Cre mice expressing ArchT ?
Recently, I discovered that there were some abnormal activities of V1 neurons in mice when archaerhodopsin(ArchT) was seletively expressed in parvalbumin(PV)-expressing interneurons.
Spontaneous local field potential of V1 in PV-Cre mice expressing ArchT were different from both in widetype C57BL/6J mice and SOM-Cre mice expressing ArchT(see the picture attached). This kind of abnormal activity seems not effect on basic response of V1 neurons to visual stimuli, that's to say, receptive field and orientation tuning could be getted.
But, after the V1 cortex was exposed in green laser for sometime, spontaneous local field potential of V1 without laser will exhibit another kind of abnormal activity(also see attached picture). That seems that laser effect V1 neurons irreversibly, inconsistent with the reversible effect of optogenetics. And visual stimuli can aggravate this kind of abnormal activity, which seriously disturbing response of V1 neuron to visual stimuli, even that you can not get the receptive of V1 neurons. It's less happened in SOM-Cre mice expressing ArchT
Is it happened in your experiments? Do you know why this happen? And how to improve the situation?
Look forward to your reply
We didn't inject virus into C57 mice. LFP of widetype C57 was just a blank control.
And now we haven't detailly distinguish the type of the cells we recorded. We assume that LFP mostly results from excitatory neurons, because excitatory neurons are much more than inhibitory neutons. Apparently, two-photon guided recording is the best way to distinguish cell types. But our lab lacks relevant instruments now.
Whole cell recording in slices is a good advice, Thanks a lot .Following
- Alan Robinson added an answer:5Do any optogenetics people know where I can get high numerical aperture optic fiber?
I've made good optogenetic fiber implants for lasers in the past using lower numerical aperture (NA) silica core fiber from Thorlabs. Recently, we've been working with LEDs which require a higher NA fiber in the 0.6 - 0.7 range. The highest NA fiber that Thorlabs carries is 0.5. Prizmatics has the right silica core fiber with high NA, but they only have core diameters of 200um & 250um.
Does anyone know a good vendor for this kind of fiber ( bare fiber, silica core, ~0.65NA)?
Can you clarify what you expect with "bare fibre, silica core"? Do you mean an oxide glass structure with all polymer coatings removed?
As Benoit points out, it is difficult to achieve numerical apertures higher than 0.24 in an all-glass structure and a pure silica core. A doped silica core with higher refractive index gives more scope for increasing the NA, but may show reduced transmission for shorter UV wavelengths. This is probably not a concern at 350 nm and longer wavelengths, but highly doped GeO2-SiO2 fibres have their own problems with stresses induced by differential thermal expansion during manufacture.
Polymer claddings are available with lower refractive index than fluorine doped or boro-fluoro doped silica, but it is difficult to find suitable materials with refractive index below 1.37 (NA 0.5). There are fluoro-polymers with promisingly low refractive index, but I don't know if they are suitable cladding materials.
Can you provide a link to the Prizmatix fibre you refer to? The only 0.63 NA offerings I found were polymer core fibres.
Do you require mechanical flexibility in the fibre? A 1000 micron diameter silica fibre is quite stiff. Could you use a section of bare fibre or pure silica rod, perhaps supported and aligned by PTFE bushes, with LED power coupled directly or from a polymer optical fibre?
Surrounded by an aqueous medium, the numerical aperture would be around 0.6, exceeding unity for the air-clad sections. Would this work in your application?Following
- Bruce Koch added an answer:3Do you have any good methods for retrograde labeling of neurons for optogenetic silencing?
I would like to optogenetically silence specific neurons by retrogradely labeling them using a viral vector. Im thinking of using an AAV-Serotype 6 virus with ArchT. Anybody knows if such a vector exists or have experience with it? Any help/advice would be appreciated
Sorry, I don't know. CAV-2 is only available from that French core - you'll have to speak with them.Following
- Fengling Li added an answer:2Is pAAV-EF1a-double floxed-hChR2(H134R)-EGFP better than pAAV-EF1a-double floxed-hChR2(H134R)-mCherry as a carrier for optogenetics ?
Some paper said that,'Clumping of a channelrhodopsin
fused to mCherry was apparent , suggesting that mCherry may not be an ideal
fluorophore for opsin usage'.So I'm considering if it is necessary to reconstruct the carrier,pAAV-EF1a-double floxed-hChR2(H134R)-mCherry into pAAV-EF1a-double floxed-hChR2(H134R)-EGFP. What's yoursuggestion?
Hi, Dr. Volker Busskamp,
Thanks for your reply and suggestion. We are going to buy some virus carrying pAAV-EF1a-double floxed-hChR2(H134R)-eYFP to apply. And I think it is a wise decision to test pAAV-EF1a-double floxed-hChR2(H134R)-mCherry by myself in my experiment as you said. So I will also try it.Thank you again.Following
- Pawan Bista added an answer:15How can I get enough fibers labelled with ChR2 to trigger light-stimulated responses from long-distance projections?
I am confused about the long-distance expression of ChR2. Recently, I injected AAV-ChR2 in ventral hippocampus of mice, After 4-6 weeks, I recorded the light-stimulated EPSC from prefrontal cortical slices. The ChR2 expression is good in vHIP, however, I had few fibers labelled to evoke blue light induced responses in prefrontal cortical slices~ Could someone help me?
I have been injecting regularly AAV2/9.CAG.ChR2.Venus ((Plasmid #20071) from Addgene for tracing as well as for exciting long range cortico-cortical as well as thalamocortical dLGN to V1 projection, which works consistently without any problem. The expression is always strong enough to get postsynaptic current just by depolarizing the axon terminals at the recording site which can be achieved just in 2 weeks after injecting the virus. I inject just 50 nanoliter in dLGN and 100 nL in corticocortical projection. I think this shall work in your case as well. All the best.Following
- 12Has anyone had problems with a channel rhodopsin AVV killing neurons?
I purchased the CamKIIa-ChR2-eYFP from the UNC vector core. This is the one from the Deisseroth lab.
My problem is that when we infected some rats with this virus and took fresh slices for electrophysiology (~5weeks post injection), it looked like almost all of the neurons at the injection site were dead under the infrared camera. There were still plenty eYFP positive projections, but almost no soma.
However, I also ran a second batch of adult animals at a separate time point. When I perfused rats 8 weeks post-AAV injection and slice/mount the fixed tissue, I can see very dense eYFP projections at the injection site (similar to what was seen with the fresh e-phys tissue). Oddly, the soma are completely devoid of eYFP (looks like dark holes in the tissue, figure attached), but that pattern looks similar to representative images I see in other publications. There is also still DAPI positive staining in the nuclei. I've been told that this is just the result of the ChR2-eYFP fusion protein moving into the terminals. This makes me think there wasn't much of a problem, at least in this batch. I've had mixed results running behavior with optogenetic stimulation.
Has anyone had a problem with cell death using AAV5-CamKII-ChR2-eYFP?
Did the injection volume vary between your animals, or between the two batches of surgeries? I've heard of people seeing vastly increased cell health at the injection site and increased axonal transfection at projection sites after reducing the volume of virus injected. While it likely depends on titer, a volume of ~30-50 nl per site seems appropriate in cortex for typical (e.g. UPenn) AAVs; not sure for your injection locations.
Regarding appearance of soma: with ChR2-fluorophore fusion proteins, I believe it's common to have a dim somatic signal since it's just in the membrane and not cytosolic, while the dendrites and axons may appear to have a more discernible signal.Following
- 4Does anyone know the distance a certain optical light can diffuse in the brain issue?
Hi, I am trying to use optogenetics to study the connection of two nucleus within rat brain. However, the distance between this two nucleus is only 1mm. Does anyone know the distance a certain optical light can diffuse in the brain issue? Thanks!
Bottom line: if you're using a fiber, and place the tip ~200 um from the stimulation target (a common distance), your target will see only ~10% of the light intensity measured at the tip of the fiber.
Another useful source, that synthesizes from a number of publications:
This includes measurements by Svoboda's group that look at the lateral spread for an LED on the cortical surface.Following
- Nicolas Grandchamp added an answer:6Difference between localization of gene expression in AAV Vs. lentiviral vectors?
We use viral vectors to deliver genes in optogenetic experiments in rats. I have recently read a claim that lentiviral vectors result in more localized expression as compared to AAV (e.g., Yizhar et al, Neuron 2011; Hirai, Cerebellum 2008). We usually work with AAVs as it is more simple (especially with regard to safety protocols).
1. What is your experience with this issue - did you observe such differences in your preparations?
2. If this is indeed the case, does anyone have an idea of the mechanism underlying this difference in the level of localization?
- In my lab we did comparisons AAV/LV after stereotaxic injection (striatum, hippocampus / rat&mouse), the results show a better spreading with aav, but better level of expression with Lenti.
- I confirm the effect of the mannitol and also after LV injection in spinal cord
- Konrad Müller added an answer:3Does someone have a reference for the (binding) response of the PhyB-Pif6 optogenetic system to all wavelength from 600nm to 800nm ?
Basically, many papers say they use 650 and 750 nm light but I wonder if someone measured some for of response curve for intermediate wavelength. (wheter in vitre or in vivo)
We have illuminated our PhyB/PIF6- based gene expression system with light of wavelengths between 650 nm - 760 nm. You can find the data in Fig. 4 of Nat Protoc 9(3):622-632...Following
- Joy Arthur asked a question:OpenHas anyone tested PPR/ LTP in chAT-ChR2 mice?
produced from crossing ai32 mice http://jaxmice.jax.org/strain/012569.html with ChAT-Cre miceFollowing
- Lief Fenno added an answer:4Does anybody know of a cheap fluorescent dye one could use for quick coordinate verification injections?
Currently in our lab, we've been using green retrobeads (Lumiphore) during stereotactic injections to verify coordinates before we inject optogenetic viruses or implant fibers. They work fine but are really too expensive for this purpose since they're intended for retrograde tracing. Does anybody have any recommendations for a cheaper dye with a similar strong florescence (sometimes visible to the naked eye)? Thanks!
Methyl Blue works pretty well, too and we usually have it laying around the culture room-Following
- Lucas Koolen added an answer:8Is anyone aware of an optogenetics system that uses expression of a fluorescent protein as output (instead of an action potential)?
I'm looking for a way to monitor the trafficking of metastatic cancer cells in a mouse. Optogenetics seems like a nicely controlled way to turn on gene circuits in one location (perhaps through a g-protein coupled receptor) and then look for these cells that have had this gene circuit activated in other locations in the body. Essentially I'm looking for an optogenetics system that would allow for light activation of a gene circuit that results in expression of a fluorescent protein. Has anyone heard of anything like this? I know many of the channelrhodopsins are tagged with GFP or YFP to allow for identification of cells that can be activated by light - but the output of this activation is an action potential not gene expression.
I believe the type of tool you are looking for is often referred to as an 'optical biosensor'. these are engineered proteins in which a light sensory protein domain (often bacterial phytochromes) are linked to an effector protein so that activity of the latter causes the former to light up (rather than the other way around: light stimulation leading to effector activity). several tools like this have been developed in recent years, but they often require substantial engineering efforts to make them meet your specific experimental requirements. here's a review that discusses recent developments in the engineering of phytochrome based optical biosensors:
- 9Optogenetics: Is it possible to use it to make an F-I curve for the neuron, with light instead of injected current? Any references?
In electrophysiology we often characterize a neuron by its spike rate output in response to a current input. This is often called the F-I curve, as frequency response (F) to different injected current (I). But is it possible to replace the injected current with light and thus determine the F-I curve using variable levels of light -and thus being able to determine the FI-curve using extracellular recordings alone? Thanks!
Adding one more point regarding direct/synaptic inputs - I believe that strong direct activation of a ChR2-positive neuron can lower its input resistance substantially, but I don't have a reference handy ... and unlike somatic current injection, in most cases this effect will be distributed throughout the dendrites as well.
Which brings me to another point: depending on your choice of objective lens (specifically, magnification), together with brain area and cell type, the exact positioning of your field-of-illumination can have a substantial effect on the effective stimulation:
In slices you'd care whether you are illuminating the somatic compartment only, or the entire dendritic arbor, or a subset of the dendritic arbor. Furthermore, since you will likely have multiple ChR2+ neurons in your slice, you would see a combination of direct and synaptic excitation - the ratio depending on how many other neurons (and their processes) are within your field-of-illumination. In slice, one way to achieve consistent illumination is to use an inverse Koehler configuration to stimulate via the epifluorescence path of your microscope - by using a low magnification objective lens and adjusting stage position, you can ensure even illumination across the full dendritic arbor (and i.e. cortical layers).
In vivo it can be more difficult to achieve even, repeatable illumination. If you illuminate the surface of the brain, most of the activated ChR2 will be near the cortical surface (mostly in L1, some in L2/3, very little below that) because of the steep decrease in intensity of blue light at greater depths (see for example this openoptogenetics.org wiki page for a collection of publications).Following
- Muhammad Aslam Baig added an answer:5How do I calculate the exact laser energy with my optogenesis experiment?
I am doing optogenesis experiment with my cultured neurons. I am kind of confusion how to get exact laser energy expose to my special compartments on my cultured neurons. For example, my 488 laser line total energy is 25 mWatts, and I used 2% to scan the specific part on my cell. How can I calculate the exact laser energy?
I suggest you measure the power of your Ar ion laser using a power meter, don't rely on the mentioned power by the company as it decreases with the age of the laser, and then place a 10% transmission neutral density filter and again measure the power at a distance where you are going to place your sample for exposure. Monitor if the power is 10% of the original power. If there is some difference it will give you the measure of the absorbance of the filter materiel. I expect you are not using any focusing lens to expose your sample. If yes then you have to calculate the spot size and power density at the sample. Area=pi r2 and Power density power per unit area whereas r=(4/Pi)wavelength.(focal length/diameter of the laser beam at the lens.
- Ashish Gupta added an answer:2How is a particular functionality of a neuron identified using optogenetics?What is the beginning approach used in this manner?
Thank you very much for answer. It helped me a lot for understanding the concept.Following
- Bruce Pratt added an answer:8Are there restrictions on non-academic research establishments, like biotechs, using optogenetic tools like channelrhodopsins?
Do optogenetic tools and constructs, like the classic channelrhodopsins, have usage restrictions that stop non-academic research labs from freely utilising them for research purposes? Thanks everyone.
Check with your legal department about "safe harbor" provisions in patent law.Following
- Lupeng Wang added an answer:5How is optogenetics cell type specific?
I am curious to know, how do optogenetics offer cell type specific delivery of opsin gene into tissues? I'll be much obliged if you take your time to respond to me.
If working with mice, the most closest ones to get cell type specific expression is to use the Cre-LoxP system. There are many cell type specific cre lines available, such as GENSAT or from Allen brain institute, then you can inject viral vector containing Cre dependent opsin into the target area to achieve cell-type specificity.
Or you can creating transgenic mice through BAC library to have opsin expressed driven under cell type specific promotor.
Injecting viral vectors containing cell-type specific promotors in WT mice might achieve the same results, but it is not very reliable.Following
- Charles-Francois Vincent Latchoumane added an answer:25What's the simplest and more efficient optogenetics setup for dual (independent) light delivery?I'm looking for an (in vivo) optogenetics setup to illuminate one hemisphere of the mice brain with one light (i.e. blue for ChR2) and the other with a different wavelength (i.e. green for eArch3.0) using different patterns of stimulation. I have got some quotes from different companies but there are too many options to evaluate and I'm still not sure about what's the best:
1) Laser vs LED based system
2) Normal LED vs miniaturized LED
3) How much power (mW or mW/mm2) do I need at the end of the cannula for efficient stimulation of these channel?
Any comments will be very appreciated.
I agree that regular LED and microLED solution do not provide satisfactory output power.
In our lab we are using 125um diameter, dispersive or straight cannula (Doric Lens) which should output >0.5mW (usually ~2mW, for both blue and green) for good behavior results and recently we found a good alternative. We thought that only crystal laser were could output >10mW at the end of the patch cord resulting in ~2mW at the end of the cannula, but this company makes LED based laser that can output up to 40mW (patch cord, I have reached up to 15mW output from a dispersive type cannula) when correctly calibrated: cnilaser.com (made in china).
It seems that those laser have a very fast response to TTL drive and provide really nice power for in vivo applications. Hope it helps.Following
Optogenetics is an emerging field of study that seeks to utilize photoreceptor proteins to manipulate neuronal cells through light stimulation. The practical applications could be far reaching including aspects of specific drug targeting in disease, direct gene targeting, and integrated fiber optics.