Fengling Li added an answer:Is 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:How 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
Has 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
Are 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?
A useful article comparing AAV serotypes from the Rumpel group (they find some retrograde labeling with AAV5): "Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain"
Does 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:Difference 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:Does 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:Has 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:Does 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:Is 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:
Amy R Nelson added an answer:Does anyone have any advice on optogenetics: Photocurrent run-down?I'm using optogenetics to measure light-evoked IPSCs in the amygdala and have encountered a problem with photocurrent run-down, i.e.,initially, 1 ms light pulses spaced 30 seconds apart evoke large IPSCs but over 3-5 minutes the IPSCs often (but not always) become smaller and smaller until they disappear. AAV-DIO-ChETA was injected at least 2 weeks before slice preparation for in vitro whole-cell recordings.
Has anyone encountered this problem? and if so, any advice would be appreciated!
Speaking as a non-expert on this topic, it is important to consider phototoxicity. You should consider reducing your light intensity or length of pulse or increasing your time duration between pulses and see if this prevents the rundown you are experiencing. When thinking about causes of rundown in extracellular field recordings possibilities include movement (of the slice or electrode), overstimulation (which I think I would explore in this case), sick slices (which is a possibility also), drift in temperature (sometime air draft can cause this) or some other unknown factor. You could confirm slice health by trying to get a baseline doing field recordings. I would wager the problem is the light though.Following
Optogenetics: 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:How 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:How 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:Are 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:How 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
Juan Roa added an answer:What'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 already asked for this kind of setup. The problem is the power :
Only 0.5-0.8 mW for green-yellow using 200 um fiber. So, maybe not enough for ArchT or halorhodopsin. Also, if you want to use 2 wavelengths at the same time coupling both to a rotary joint (for in vivo experiments) like the Doric FRJ 2x2 FC-FC, the light at the end of the cannula will be even lower (aprox 20% lower).
This is why I discarded LEDs. Is the cheaper option but maybe not very useful for some applications.Following
Hong Zhan asked a question:Is it any literature about optogenetic (channelrhodopsin/halorhodopsin) and SPT in cultured cell or tissues?
Is it any literature about optogenetic (channelrhodopsin/halorhodopsin) and SPT in cultured cell or tissues?Following
Jocelyn Seemann added an answer:How or where through NIH can you get clozapine-n-oxide (CNO) for free (or at a reduced price)?It was mentioned at the optogenetics and chemogenetics short course at SfN that it was possible to attain clozapine-n-oxide (CNO, the DREADD ligand) for free or at a reduced rate through NIH. Is it through BrIDGs at NCATS (http://www.ncats.nih.gov/research/reengineering/bridgs/bridgs.html)? I got the sense at SfN that the program was available for any/most researchers, not just preclinical researchers.NIDA Drug Supply Program (DSP) is now producing CNO, which we have successfully applied for and received! More info re ordering: http://www.drugabuse.gov/ordering-guidelines-research-chemicals-controlled-substancesFollowing
Elwood Siagian added an answer:Could optogenetics provide a new interface between electronics and biological nervous systems?The control of robotic prosthesis via signals from the nervous system has reached an impressive level in recent years. However, the feedback of signals from the prosthesis back into the nervous system, e.g., to provide touch sensation, remains a much more challenging problem. Could methods of optogenetics provide an alternative interface in this context?Hi Jochen, that is a very interesting question and I agree, we may have to redefine these sensations at least for those wearing the prosthetic. It sounds promising and the clinical applications would be tremendous!Following
Braxton A Norwood asked a question:Experience 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?Following
Aaron Mercer added an answer:What novel technologies do we still need to develop for neuroscience research?The last decade has seen fantastic progress in the emergence and development of research tools to study neuroscience and interrogate normal and diseased brain function in vitro and in vivo; tools like iPS cells, optogenetics, CRISPR/cas9 etc. I'm interested to hear your opinions about what tools and technologies you think we still need to discover and invent to uncover more of the mysteries of neuroscience and the big neurobiology questions of the day?In regards to Jonas' comment, it would be great if we could have CLARITY-like living brains to observe circuits in intact brains in freely-behaving animals. We have the tools to look at all kinds of live-cell physiology in neurons, but no capacity to do it in deep brain structures in intact animals very easily.Following
Lief Fenno added an answer:Can channelrhodopsin and halorhodopsin be expressed in the same cell using Cre/Lox system?I am trying to figure out how to breed my mice in order to express different rhodopsin channels in the same cell. I have one line of mice that express Channelrhodopsin, another line expressing halorhodopsin and a 3rd line cre recombinase mice. I was wondering if I could breed the 2 different lines of mice which express the different rhodopsin channels prior to breeding them with the cre recombinase line. I am hesitating to do so because I think there might be homologous recombination between the 2 lox sites (which are both in locus Rosa 26).
Thank you!Shoot me an email and we can chat email@example.comFollowing
Jonathan T Ting added an answer:Can channelrhodopsin and halorhodopsin be expressed in the same neurons?I am trying to find a way to induce the excitation and inhibition on the same set of neurons that are relevant to a particular behavior in rodents. Can you express both of these opsins in the same cell and activate them alternately and simultaneously?You can express both in the same cell using Cre/loxP. You would ideally have both genes in a single double-floxed cassette e.g. viral vector with DIO design. You could then inject that virus into the brain of a mouse or rat with cell type specific cre recombinase expression. That would be the obvious way. If you have ChR2 and NpHR on separate expression vectors, e.g. two independent DIO viral vectors, this could in principle cause some issues with undesirable recombination events when virus is co-injected into a Cre mouse, but I have not tried this.Following
Juan Roa added an answer:What is the best serotype of Adeno-associated virus (AAV) to infect hypothalamic neurons?I want to deliver AAV to infect specific neurons in the hypothalamus. I have been checking some papers and people use mostly AAV2 and AVV8 but I'm not sure which one will work better in terms of spread of infection, time, etc. Any help would be greatly appreciated. Thanks in advance.Thanks Valery!Following
Satya Sharma added an answer:What is the scope of the application of optogenetics in human subjects?As being declared method of the year 2010 by nature but requirement of expression of foreign protein and a high grade of invasion, I am really curious (not criticizing) over the potential of Optogenetics in Human subjects. Workers at Salk institute have come up with expansion of codons, but problem remains more or less same. I want to know is there a scope of modification or we are going to have pure human lines with recombinant brain (ethical challenge) or this method will remain limited to murine and other lines?Thanks Max but, its not only about infecting with virus for gene delivery, its about uniformly infecting, producing pure lines i think that is the major challenge...Following
Jonathan T Ting added an answer:How can I optimize the expression of Channelrhodopsin for optical stimulation in the brain?My targets are the VTA, PFC & NAc in the rat, using HSV with either CamKII or EF1a as promoter.
(Commercial vectors were procured from MIT)These are all great answers. I have had good luck with AAVs produced by UNC Chapel Hill as well. I recently worked for 2 years at MIT and heard mixed stories about the success of the LT-HSVs from Rachel Neve...I think this will be very dependent on the experimental context but is certainly worth trying out. It is worth noting that Ian Wickersham is also now running a group at MIT (Genetic Neuroengineering Group) and has expertise in Rabies tracing among other cutting-edge genetic labeling strategies. You should also look at the large catalog of AAVs for optogenetics research on the U Penn vector core site as a more cost effective way to get started:
The EF1a promoter is most useful for Cre dependent expression strategies (e.g. DIO designs), whereas the CaMKIIa or hSyn1 will work well for direct expression in WT animals). Lief and Anton are quite the experts so it would be worth looking over their published work on the topic.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.