- Peter Smetacek added an answer:Do you have pictures of the experimental setup for electrophysiological recordings of the moth's auditory circuit?
Based on my reading of numerous papers, I have created a mental picture of the experimental setup used for electrophysiological recordings of the A cells excited by ultrasound stimulus. I need to check my understanding though.
Would you have, or would you be able to obtain any pictures of experiments?
I am specifically interested in close ups that may show most of the following together: the sound source, the microphone, the moth, the mounting plate, the electrodes.
Are you in contact with Lutz Thilo Wasserthal? He did some experiments during the 1980s and 1990s that might fit in with what you are looking for.Following
- Marcel Ruiz-Mejias added an answer:Which is the best anesthetic agent to use for electrophysiological recording?
Which is the best anesthetic agent to use for electrophysiological recording namely EEG and ECG?
Yes, you can use chloral hydrate for rapid surgeries. It presents fast pharmacokinetics and the awakening is pretty easy. I was not aware of its damage to abdominal organs, but you may use a SC infusion pump for a long-term anesthesia.
In the lab of JM Delgado and A Gruart they are using it for quick surgeries for chronic LFP experiments, as far as I know.Following
- Eva Maria Kugler added an answer:Can someone suggest to me the best media combination for DRG neuron in culture (at least 7 days) where TRPV1 channels will remain active?
I tried to culture DRG neuron with Neurobasal A, B27, L-Glutamine P/S and 50ng/ml NGF for electrophysiology purpose. My cells do not look healthy. I would appreciate if you input your protocol. Thanks.
I used Medium M199 (125ml) + Glucose (0.625g) + Penicillin/Streptomycin (1.25 ml) + mNGF (50ng/ml) + 10% FBS. The cultured DRG cells responded to nicotine as well as ATP. I recorded the responses with calcium imaging and voltage-sensitive dye imaging. But I never looked at TRP channels in the culture.Following
- Nathanael Yates added an answer:Is that right methodology that recording EEG and ECG in anesthetized rat?
Is that right methodology that recording EEG and ECG in anesthetized rat? which is the best anesthetic agent to use for electrophysiological recording in rat?
Unfortunately any anaesthetic is likely to alter cortical synchrony and responsiveness. In a way that's what anaesthetics do, alter cortical activity resulting in unconsciousness...Following
- Pellegrino Lippiello added an answer:Are there Differences between (-)-Bicuculline methochloride and (-)-Bicuculline methiodide for electrophysiological recordings on cerebellar slices?
I would like to know if there are any important differences between these two molecules because the price is extremely different but ((-)-Bicuculline methiodide price is very cheap) both are water soluble.
Thank's you all for your reply! So i will buy (-)-Bicuculline methiodide.Following
- Filip Melinščak added an answer:How we can acquire the EEG signal that is responsible of hand movement?
How many electrodes that should we us to control the movement of the right hand joints (shoulder, elbow and wrist) by EEG signal,e ? What are these electrodes?
I'm not sure what you exactly mean by "determine this electrodes"?
Usually people use caps to fit electrodes to the scalp, and the layouts of the caps are usually provided by the manufacturer and comply with the 10-20 system I linked in the original answer. If you have a cap, look up the layout for it and it should have positions C3, C1 and Cz.
If you don't have a cap, you can determine the positions using a measuring tape and few anatomical landmarks (see the link).
The C1 position is not defined in the original 10-20 system, but it is halfway between Cz and C3.Following
- Kevin S. Jones added an answer:Would the electrophysiological response change due to the flourescent protein tagged to the channel rhodopsin?
For neurons transfected with any variant of Channel rhodopsin, would the type of tag affect the electrophysiologcal response in any ways? For example, would the electrophysiological response change if ChR2 is tagged with GFP or YFP? Most literature uses YFP tagged ChR2. Is there a particular reason for the same?
In my hands I did not notice any obvious influence of the color of the tag, but I was not specifically looking. I'm not sure your question has been rigorously tested, but you may get the information you're seeking in the excellent review article that was in Nature Neuroscience called Ultrafast Optogenetics Nat Neurosci. 2010 Mar;13(3):387-92.Following
- Abudukeremu Kadier added an answer:Does anybody know why the most of MEC/MFC studies tested are at applied voltage range of 0.2V to 1.1V ?
I am working on single -chamber MEC/MFC studies , from the literature review I found that all MEC/MFC have tested at applied voltage range of 0.2V<Eap<1.1V. Are there any reasons for using that range of applied voltage. How about <0.2V or >1.1V?
Dear Anirban Kundu,
Do you have any reason for saying that? and do you know any links or publication about this matter?
- Serguei N Skatchkov added an answer:Why is my baseline drifting dramatically after offset and before whole-cell patch with the Cs-gluconate internal, but not the K-gluconate internal?
There is no other difference besides K+ and Cs+.
THANKS! I wait
DO not worry, you are correct, my first name is Serguei and last is Skatchkov
- Yoram Oron added an answer:For electrophysiology and calcium imaging, does some sore of ATP chelator exist?
I need to quit ATP of the extracellular medium but not by hydrolysis or inhibiting its synthesis.
Hi there. The best way to chelate your ATP so it does not chelate your free calcium would be an excess of magnesium chloride. Since your total adenosine nucleotides should be around 5 mM, 5-10 mM Mg should do the trick without affecting calcium-probe interactions too much.Following
- Mau Yip added an answer:What are the best electrodes available for in vivo LTP induction?The rats would swim in water maze, I need electrodes to penetrate different layers of hippocampus, which probe to use?
Can you give me some suggestion how to make recording electrode and connect it with headstage? Thanks!Following
- Rizwan Qaisar added an answer:Why don't I see hypertrophy of plantaris after ablation of synergistic muscles?
I removed gastrocnemius and soleus muscles from two months old WT mice (n = 4) but apparently two weeks of overload had no effect on plantaris size, compared to sham-operated mice. The wounds healed nicely and the mice were active so inactivity is out of the question. All mice are young and in growth phase. I identified plantaris correctly next to the bone. Literature reports 50-100% increase after two weeks of overload but I see no change in CSA.
Any suggestion or explanation ?
Dear Willem and Ferdinand,
Many thanks for the suggestion, I guess this might be the problem. I have been removing much of the gastrocnemius all the way close to the knee, and I might be cutting nerve or blood supply to plantaris.
Online literature and atlases do not give much tracking of the blood/nerve supply to plantaris. But I feel the problem might be solved, I will do another mouse later today to see if it responds better.
Loads of thanks,
- Marta Załęska-Kocięcka added an answer:Young Investigators Research in Cardiology, Electrophysiolohy - Are you interested in collaboration?
I was just wondering if young investigators from Europe and the US would be interested in collaborating on multicenter clinical trials in the field of cardiology and electrophysiology. This may help those at the beginning of their career establishing them as potent partners in clinical cardiology research.
Looking forward to hearing your thoughts.
Dear Valentina, I work in cardiac intensive care unit and I am interested in echocardography. In my institute there is EP unit. I would be glad to participate in the project.
Best regards MartaFollowing
- Diego Fernández added an answer:How can I perform fEPSP in mouse hippocampal slices without using carbogen and recirculating the ACSF?
In the near future I must perform a series of experiments with a compound which is highly sensitive to the mechanical movement induced by bubbling the ACSF. So we deliberatly need to remove the carbogen in our experiments.
What I have tried so far is the addition of 30 mM HEPES to try to maintain the pH stable. So I tried experiments using the regular ACSF + HEPES, and I adjust the pH to 7.3 - 7.4 just before the recording. However, the slices are by far not as good as with carbogen. I thought about saturating the ACSF with carbogen just before the experiment, but then the slices are not that stable. Any other idea?
Furthermore, I don't have too much quantity available of this compound, so we probably need to recirculate the ACSF in these experiments. Do you know how much this procedure can compromise the recording?
Does anyone have any suggestion regarding these two technical challenges?
Thank you very much for your attention.
Thank you all for your contributions.
I am still struggling to perform this experiment. I think I can get good recordings for around 2 hours using a big Petri dish (to create a big surface for oxygen interchange). I try to create a oxygenated atmosphere inside this Petri dish, but avoiding to directly bubble the ACSF. It seems to work.
- Eric Dubuis added an answer:Are there any critical steps for patch clamping of mouse bladder smooth muscle cell?
Recently, we've tried to do patch clamp recording of mouse bladder smooth muscle cells. We followed some procedures from some articles, however, we are in trouble with doing this. Although we got many smooth muscle cells from enzymic dissociation of bladder, much of them seem not healty enough for patch clamping experiments. Many of cells did not attach to the plate and cells that attach on the plate are moved by the patch pipette. And making seals or rupturing cells are very difficult.
So, I wonder whether there are critical steps for for patch clamping of mouse bladder smooth muscle cell.
please help me out
Hi there,one question, did you culture the cells before patching them, for how long ?
Right after isolation the smooth muscle cells will look shiny in the shape of a spindle.
If cultured however they will look flat with a dense circle in the middle (a bit like pan fried eggs)
If you try to patch the cells immediately after isolation, I would recommend you to let a drop on a non coated glass chamber. when you patch the cell, after you make the first contact with the cell and it adhere to the pipette tip (typically when the seal get up to 200-300MegaOhms), lift the cell off the ground so it is in the liquid phase and then finish to seal them. That will allow you to avoid loosing the cell because it is moved by your pipette while sealing. With this method it doesn't matter if your cells are adherents. Also you have less risk to loose the cells when rupturing the membrane.
You said that the cells are moved by the pipette, I'm not sure it the movement is intentional or result from a drift from your manipulator. the manipulator need to be very steady, check if you have any drift and fix it. That is very important especially if you cultured your cells.
If your cells have been cultured they look like pan fried eggs (as I said above) and should be strongly adherent. Non adherent cells are dead in this case, don't use them. When the cells are adherent it is essential that your pipette is very steady (check the drift), go for the flat cells in this case, remember that they are very flat so be extra gentle. One technique which can help is: As you approach the cell (very close) apply a positive pressure through the pipette as you move it until you almost touch the membrane. You should see a depression in the membrane cause by the efflux of intrapipette solution. At this point stop the pressure,the membrane will immediately come back and touch the pipette (sometime sealing by itself) and you can apply a gentle negative pressure. As the cells are spread out on the surface, it is more difficult and take more time to get a seal and you have to avoid strong negative pressure as the membrane rupture easily.
Your protocol for isolation looks ok for me, (although the collagenase step seems a bit short, but you use a strong collagenase). The oxygenation is to keep the cells alive and is necessary for some cell type (some are starving for oxygen) even if you use HEPES. For smooth muscle cells however that's not crucial especially with your protocol which has short incubation times.
Make sure that your intrapipette solution is slightly more acidic than your bath solution (pH 7.2-7.3 is good) and slghtly hypo-osmotic (~10 mosmol) compared to your bath solution. Often if your pipette solution is hyperosmotic compared to your bath solution this result in problems to achieve a seal.
Another advice is to switch the bath solution to a solution containing ~20 mM bivalent cations like BaCl2, CaCl2 or MgCl2 immediately prior to do the seal. Some membrane coat with bivalent cations and sometime it helps to facilitate the seal formation and stabilize it with smooth muscle cells. As soon as the seal is done and you have your Giga, revert to your regular bath solution.
Hope this will helpFollowing
- Jonathan T Ting added an answer:Can anyone provide advice on purchasing a vibratome for patch-clamp electrophysiology?
I'm looking for advice on purchasing a vibratome for patch-clamp electrophysiology. We'd be making cortical and hippocampal brain slices. Currently we use a Vibratome Series 1000 which works remarkably well for its age, but it could give up the ghost at any second.
I realise that z-axis deflection is important and that the Leica VT1200S is the top of the range instrument. That said it seems rather expensive and we won't be doing fancy dendritic recordings etc...
Does anyone know what happened to The Vibratome Company?: is the Vibratome 3000 Plus still made?
Has anyone tried the pelco easiSlicer: http://www.tedpella.com/easislicer.htm?
Any thoughts or recommendations would be highly appreciated.
Hi, and thanks to all for the positive comments. Joseph--I hope you got what you need. I really enjoyed your recent study in Nature Neuroscience on Arch and NpHR.
Kevin, I think you can easily do any slicing angle with the Compresstome models, but you need to work at the blocking and mounting technique to make it reliable. I often use a special angle for hippocampal CA1 slice preparation from whole brain blocks in order to slice both hemispheres simultaneously, and have found this to work very reliably. In fact, the agarose embedding makes it even easier to maintain the intended angle of slicing, assuming you are able to securely glue the base down. I also did oblique angles for cortico-striatal recordings, and in my experience it is more about how reliable the human is in blocking at the correct angle rather than a limitation of the machine. The small size of the specimen holder for the Compresstome may prevent you from slicing the whole brain or whole hemisphere, but there is now a larger specimen holder available that might help improve that, if required.Following
- Marco A Delpiano added an answer:What osmometer should I use for electrophysiology? And how frequently do you make your patching solutions?
Hi guys, I am a beginner patch clamper and the only one that does ephys in my lab. Hence, we do not have all of the necessary equipment including an osmometer. I am currently going to another lab to check the osmolarity of my bath and internal solutions. It is not very far of a trip, but I am hoping to make fresh bath solution everyday so this becomes a bit more convenient. I am patching on embryonic hippocampal neurons and some other people recommended me to use fresh solution everyday. What is your take on this? Do you have a good brand of osmometer that is affordable for a small lab?
Thanks for reading! :)
I used a Semi-Micro Osmometer from KNAUER (http://www.knauer.net/de/produkte/produkttypen/osmometer.html). Different pipette solutions were prepared with adjusted pH and osmolality in 100 ml Merck bottles and stored in the freezer. Each solution, depending of the experiment I wanted to do, was warmed every day early morning in a warm bath (28 Celsius). Bath solution were prepared in 1-liter bottles and stored at 4 grad Celsius in the refrigerator. Remember to control every day pH and osmolality, they are quite critical for patch-clamp recording or any other in vitro experiments.Following
- Gustavo Campos Ramos added an answer:Is macrophage polarization tissue type dependent or independent?
I would like to know whether macrophage polarization and activation highly depend upon tissue type or it is just based upon the type of stimulant that mediates the activation irrespective of the tissue type.
I am analysing macrophage transcriptome data of different tissue origin. I am trying to understand the macrophage polarization in different biological conditions ( disease, treated with endogenous and exogenous stimulants ). Can I remove tissue specific biases in this case ??
That's a very interesting question indeed, but I am not sure if you'll find a definitive answer.
I can only tell you from my experience. I work with cardiac macrophages/ leukocytes and they are quite peculiar. Resident macrophages are naturally polarized towards M2, but they don't express arginase, for example. I recommend you the following paper from Pinto et al (2012).
- Jason Ramey added an answer:Does the use of lidocaine gel (vs plain lubricant) prior to foley insertion reduce trauma/bleeding in male patients who require heparinization?
I currently work in an Electrophysiology Lab where we routinely Foley patients requiring left sided heart procedures that necissitate the use of heparin. I am constructing a paper and developing best practice guidelines and looking for research, with minimal results. Any ideas on such historical research are appreciated.
The best study I found on the topic of lidocaine and catheterization was performed by Adam Singer MD from the State University of New York--randomized, double blind. Though they did use a convenience sample. It did not address the role of heparin--only pain reduction.Following
- Huong Ha added an answer:Is it possible to use resistance rather than osmolarity to test the suitability of solutions for electrophysiology?I can't find an equation giving a relationship between the two measures - we can easily measure resistance but have limited access to an osmometer.
Hi guys, on a slightly related topic. what kind of osmometer is your lab using? I would love to have your suggestion of what osmometer is reliable and affordable in order to get one for my lab. Since I am the only main person doing electrophysiology in the lab, it is quite not cost-efficient to get the fancy one like Advanced® Model 3300 Micro-Osmometer. Thanks all!
- Vito S Hernandez added an answer:Is there a way to audio monitor an electrophysiology signal using Clampex 10?
I am obtaining Whole cell current clamp recordings through an axoclamp 2b, Digidata 1550, and Clampex 10. However I would love to hear the spikes, using the PC speakers. Is there a function in clampex that I am missing?
Thank you for all your answers
In fact I though that an amplifier and a pair of speakers conected to the Signal output via a BNC-Audio jack adapter could be the solution, however If you solder a BNC cable with a Audio 3.5mm Cable, it is possible to connect directly the BNC output (Monitor) to the Mic jack in the PC, and in windows 7, there is a mode were all sounds received via the Mic are re-directed to the system speakers, that worked for me.Following
- Ewa D Zarnowska added an answer:Can anyone help with whole cell patch clamp stability?
I recorded mitral cells using whole cell patch clamp. In order to carry out my protocol, I need the whole cell configuration to remain stable for relatively long periods of time in voltage clamp. For some reason, usually 1-20 minutes after entering whole cell configuration, the leak current abruptly increases by about tenfold and becomes very noisy - essentially forcing me to drop the cell. Rarely following a short wait the cell would "heal" and I could carry on, but this is usually not the case. The abrupt nature of this phenomenon leads me to the conclusion that it has to do with the seal stability, rather than some deterioration of the cell itself. Because this phenomenon is very variable (sometimes I could hold the cell very stable for >60 minutes), it is very difficult to test solutions. I tried experimenting with the pipette properties (wall thickness, pulling paradigm, fire polish), but nothing seemed to work. The only thing that may have an effect is the clamping voltage - it seems that keeping the cell very hyperpolarized (-70mv - -80mV) for long periods of time increases the frequency of cell loss.
This makes my work very frustrating - any ideas would be greatly appreciated.
I went back to your first post. If your goal is to record in voltage-clamp mode I would add some Cs+ to block potassium leak current. I see that you do not have any EGTA or BAPTA to buffer calcium influx to your cell. Also it is quite common to use QX-314 to prevent direct activation of the cell. These may not be exactly what you are looking for because I do not know exactly what your experimental goal is. In the end if it is possible ask for an aliquot of working internal solution from your friend and try it out.
There are so many things that may go wrong with internal. The ingredients may be contaminated in their original bottles. Thus, it is a good practice to have those separate from ingredients that you use for your ACSF. Also it is a good approach to buy new ingredients every year. These precautions should be taken under consideration especially in situation where there are several experimenters in the lab.
Last thing that comes to me is asking you to double check osmolarity of your recording aCSF. Few year ago I witnessed a long-term recording with a huge leak. It turned out that ACSF was hypoosmotic with 150 mOsm.
Please let me know if I could be of more help,
- Imre Farkas added an answer:Can anyone direct me to a good protocol and practical suggestions on how to perform loose patch?
I would like to patch neurons using this technique. I have read several protocols on how to do so, but would like more practical details...
Oooups.... cultured cell.... Well, then try the nearest cell first. If you achieve Gigaseal on it, remove the electrode from the surface by applying positive pressure then try the next cell with the same electrode. In that case the rim has already become dirty, so you have no chance for the Gigaseal. Sorry, I have no better idea...
- Wei Y added an answer:Where can one get hands on training in In-vivo electrophysiology (Recordings from dorsal horn neurons)?
Looking forward to your help.
I‘m recording at CA1 region in vivo.Get the parameters and practise,then you can make it.The problem is neurons layer is more thin,you need be careful.Following
- Huong Ha added an answer:Can intracellular solution influence access resistance?
I use Cs based intracellular solution. I found that the access resistance gradually increase after breaking into the cell. The figure is attached.
I also used other people's ICS to test. And the Ra changed a little during recording.
So, I have some questions to ask.
1. Can ICS affect Ra? Is there anything wrong with my ICS?
2. how to prepare ICS?
3. What should I do to avoid Ra increasing?
I actually do not have infra-red optics. My scope use the Hoffman Illumination objective, which creates a "fake" DIC image quality. I could, however, sometimes see that the cells looked funny (wrinkle membrane, visible nucleus) after being patched. Yet this phenomenon is not always there.
You are absolutely right that the AMPA currents in my culture has very fast rising kinetics, and the amplitude that I could detect is somewhere from 6 pA - 40 pA.
Nice to hear that your access could improve over times with that osmolarity pair! I will try this approach.
For the side question, I actually do not know of any supplier of CsGlu salt. :| I am also using CsOH and gluconic acid to make my internal. There is something about this salt that makes it difficult to crystalize, I think...
Thanks for the help!Following
- Hansika Kapoor added an answer:Could you recommend literature reviews on Psychopathy or Antisocial behaviour and EEG studies?
I'm looking to review literature in the area of psychopathy and antisocial behaviour, studied using electrophysiological methods like EEG. Would appreciate some suggestions of Review papers.
@Nicola: Will do that, thanks for the suggestion!
@Assia, Fernando, Hewig, Tomas, Eliyahu: Thanks so much! Will have a look at all the articles :)Following
- Saak V. Ovsepian added an answer:Can an inhibitory axo-axonal synapse inhibit a propagating action potential along an axon or does it decrease the probability of initiating one?As neurotransmitter release is a calcium-influx dependent process, if an axo-axonal synapse hyperpolarizes the cell in general and not just a segment of the axon, a complete hyperpolarization and silence of postsynaptic cell synapse is possible. If not, then an excitatory input at a downstream segment of the axon might trigger an action potential that will lead to neurotransmitter release with a failure of possibility of back propagation or c-fos expression in cell body.
Yes, it can. Activation of GABAA receptors are known to shunt the impedance of axons, which can lower the amplitude of AP and its safety factor of propagation. This ultimately may lead to propagation failure. For comprehensive reviews, look early papers and reviews by Eccles, Rudomine and others...Following
- Edward J Tehovnik added an answer:Can brain-machine interfaces be evaluated quantitatively using information theory?
Brain-machine interfaces have become important in rehabilitation with the goal to restore motor function to paralyzed people. The topics discussed in the lecture include: (1) the bits of information generated by a brain-machine interface signal, (2) the superiority of a brain-machine interface signal using single cell recordings versus electroencephalographic recordings, (3) the limitations of including more neurons for generating a brain-machine interface signal, (4) plasticity and brain-machine interfaces, (5) the selection of a neural code as implemented by brain-machine interfaces, (6) the significance of body movements while using brain-machine interfaces, and (7) the role of vision for brain-machine interfaces. During the question period the issue of using information theory to assess studies in behavioural neuroscience is discussed.
Specific topics covered by the seminar: information theory, systems neuroscience, neural prosthetics.
Full lecture as delivered at the University of São Paulo on August 29, 2014:
Dear Ardalan, We have just submitted a paper on the topic and once it is published we will share the contents with you and get your feedback. Thank you for responding to the question. Ed Tehovnik.Following
The study of the generation and behavior of electrical charges in living organisms particularly the nervous system and the effects of electricity on living organsims.