- Glenn Watson added an answer: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!
See the following article concerning light diffusion in different brain brain regions of mouse;
See recent work by Ed Boyden's group as well addressing this issue;
- Elwood Siagian 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've been successful with using AAV2/5-CaMKIIa-hChR2(H134R)-EYFP from UNC. I injected only 2ul of vector construct (4x10e12vg/ml) into the rat PFC. After 6-8 weeks I stimulated PFC while performing in vivo recording of single units and LFP in the midbrain. Depending on which fibers/soma you intend to activate, your stimulation protocol will make a difference. Good luckFollowing
- 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?
Lentiviral particles don't spread well after stereotaxic injection into brain because the particles are relatively large (~50nm if I recall correctly). In contrast, AAV particles spread more readily due to smaller size (~20nm). As mentioned, it has been stated that some AAV serotypes spread better than others in brain, for example, AAV5 is reported to spread exceptionally well when injected into striatum. Lastly, pretreatment with i.p. mannitol to your animal about 15 min ahead of viral injection has been reported to aid in spread of viral particles in the brain.Following
- Benjamin A Suter added an answer: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
- Seyed Mohammad Abedirad 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!
I think curcumin is good choice, an intense green fluorescence- (5050560nm).Following
- Artemis Llamosi 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)
I understand these 650 and 750nm don't come from nowhere but still, I don't think such round values are biologically grounded. Also, most standard light sources won't give you exactly a sharp 650nm or 750nm emission... Has anyone published such kind of "wavelength calibration" data ?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
- Simon Cork 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!
Is it definitely run down and not an artifact of loss of access to the cell? If it is run-down I would expect it to happen all the time, especially since you suggest the evoked current decreases. I've seen evoked action-potential rundown before, but this was in the absence of any reductions in evoked photo-currents and was restored after a few secondsFollowing
- 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 firstname.lastname@example.orgFollowing
- 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
- 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
- Jacek Debiec added an answer:Do Oxytocin neurons have projections to Amygdala only to CeL and not to BLA, in order to regulate fear responses?What is a role of other neurons in Amygdala like BLA other than CeL, in which inhibitory networks get excited by Oxytocin to influence CeM to change fear responses?Some authors discussing the presence of OT-ergic neurons in the LA refer to the paper by Sofroniew (1983). However, in his original paper Sofroniew reports the presence of OT-ergic projections in the CeA, CoA, LA, BA altogether without distinguishing among the nuclei. That may have caused assumptions that the LA and BA have OT-ergic fibers. To my knowledge it has not been really confirmed.Following
- Andrés Martín-Quirós added an answer:How effective and practical are optogeneic tools for controlling protein expression in microbial systems?Optogenetics tools, such as fusions of light-absorbing domains with protein 'effector' domains, should in principle allow light to be used to control protein expression in bacteria and archaea. Is this practical and useful compared to other methods with current technologies?From my point of view, "opto-" techniques are useful when studying processes that are fast, dynamic and/or very localized. This kind of studies would benefit from the enormous spatial and temporal resolution of manipulation by light. Among them, I underline cell motility, protein complex dynamics, cytoskeleton dynamics, intracellular traffic, studies on organelles and of course neurotransmission, for which optical tools can be less invasive than the rest of the available techniques.
Studies on processes with time frames of hours to days are also possible, but won't benefit so much from the properties of optical control, since they can be addressed with simpler methods. In this case, the cost of optical techniques (both in equipment and technical complexity) can outweigh their benefits.
When it comes to protein expression, I think that applying optical control would make sense if one is trying to dissect expression in different parts of a single cell or if the control is applied at the level of translation which, with proteolysis and sequestration, would be the mechanism for fast changes in protein concentration.
Note that I speak of optical tools in general rather than optogenetics alone. I myself work on the approach of optopharmacology, that is, the development of optically active molecules able to produce the control effects of optogenetic tools without the need for heterologous overexpression in order to address key questions in "native" systems.Following
- Mir-Shahram Safari added an answer:What is the best strategy for doing two-photon targeted in vivo double whole-cell patch clamp in mice cortex?Patch-clamp is the gold standard of neurophysiology, applying this technique in in vivo animal opened a new area for understanding inputs and outputs of neurons in physiological conditions, during past decade a lot of papers in nature, science, neuron and nature neuroscience published using this technique.
Also using dual whole-cell patch clamp for finding connection between different neural subtypes established very well but I am doing that in vivo mixed with optogenetics.Thanks Gregorio, I improved targeted in vivo patch to make whole-cell in single patch configuration, success ratio is fine, that was reason that I thought about targeted paired in-vivo whole-cell, actually I am doing that in paired configuration also but moving of one pipette hurt second neuron in the tip of another pipette and making good whole-cell in both (with low Rs appropriate for recording uIPSC or uEPSC in both) is extremely difficult.Following
- Athena Akrami added an answer:DREADD 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!Dear Dimitri, Thanks a lot for the references.Following
- Sungmoo Lee added an answer:Two point mutation in WPRE region of a 'pLenti' vector would prevent the expression of the protein ?I am doing research relating to optogenetics so I am trying to transduce primary neuron with the protein that I am interested in.
The protein I am talking about is sub-cloned into a 'pLenti' vector. After the transduction I checked the eYFP fluorescence but there was none. So I ran a full sequencing to find out any mutations. Gene insert had no mutation but WPRE which follows the gene insert in 3' region contains two mutations. WPRE region is called Woodchuck Posttranscriptional Regulatory Element and it is known to help the expression of the protein.
Would two point mutation in WPRE region affects the expression level of the protein that I am interested in?Dear Dr. Schloendorn,
Thank you very much for your answer.
Yes, not that I intended, but I happened to run sequencing for the WPRE region several times and the two point mutations were consistent every time.
Your comment on 'ribosome binding site' and 'IRES' is unfamiliar to me so I guess I should start find out what that is.
While lentivirus packging I never looked it up so I think I should try to check the fluorescence while lentivirus is being made in next time.
One possible explanation I thought is that the peptide I attached behind eYFP might have influenced the stability of the whole protein somehow and turned out to be nonfluorescent maybe..
I think I should try it again.
I believe I can look into it with different view in this time.
Thank you Dr. Schloendorn!!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.