- Barbara Niemeyer added an answer:1Could anybody give us any basic tips how to determine TEER with an Ussing Chamber?
We just started to use a ussing chamber system with cell culture transwells in our laboratory (U9500 Warner Instruments) and do not really have any experience in that field so far. Is there anybody who could give us basic advise what is crucial for such experiments? Assembly of Chamber, Electrodes+Agar bridges etc is not the problem, but unfortunately we do not know if our results are plausible or not.
I attached a picture of what we recorded in one of our last experiments. The lowest two Graphs are the calculated membrane resistance. Unfortunately we do not have any idea why the voltage traces look so different. It would be great if we could get some advice what could be our problem.
I would suggest to contact Dr. Jens Leipziger at Aarhus University in Denmark, who has profound experience using hte Ussing chamber.Following
- 1How do I place the stimulating electrode in mouse contralateral dorsal hippocampus to activate commissural CA3 synapses on the ipsilateral CA1?
Dear fellow researchers,
In the mouse, to study in vivo Schaffer collateral - CA1 pathway LTP, I want to stimulate the Schaffer collateral pathway from the contralateral side and record field potential responses from the ipsilateral dorsal CA1 stratum radiatum (stereotaxic location: AP: -1.46, ML: 1.0). I considered two contralateral stimulating locations:
1) the location homotopic to my recording location in CA1 (AP: -1.46, ML: -1.0, DV - at stratum radiatum) and
2) the pyramidal cell layer of CA3 (AP: -1.46, ML: -1.5, DV - at CA3 pyramidal layer).
Your input in choosing one of the two above is really appreciated because I think both options seem to have some issues.
Option -1 (stim electrode in CA1 s. radiatum) - this likely activates two pathways - a) the Schaffer collateral path ipsilateral to the stimulating side, which then antidromically activates contralateral CA1 and also antidromically activates CA3 on the stimulating side which may then activate contralateral CA1. b) the CA1-CA1 associational pathway - this could happen because the electrode might stimulate the CA1 pyramidal cells too. My question is which of the two pathways (a & b) contribute more to the evoked responses?
Option-2 (stim electrode in CA3 pyramidal layer) - stimulating from this location will orthodromically activate CA1 cells on both hemispheres. Hence, this for sure will activate the Schaffer collateral I am interested in but will also activate CA1-CA1 associational pathway. Do you think the Schaffer collaterals will contribute more to the evoked responses in this case compared to option-1?
Which option is better in general considering other things I haven't mentioned here?
Note that I do not want to place stimulating and recording electrode on the same hemisphere for technical reasons.
You managed to put a quite straightforward question in a complicated way - ) What you would like to do, as I understood, is to measure the LTP of commissural inputs to CA1 neurons of the dorsal hippocampus. And you want to know where to place your stimulation electrode.
There are numerous papers on this topic and best will be for you to check them and read some of them as there are details that you need to be aware. As a starting point I would suggest to check the paper by Bliss, Lancaster and Wheal in JP (see below). I hope this will give you some background and will serve as a good starting point.
http://www.ncbi.nlm.nih.gov/pubmed/6620191. Hope this will set you on the right path.
- Alexander Cerquera added an answer:2Is there a procedure to represent a multi-channel EEG in terms of neuro-spike time intervals?
Is there a procedure to represent a multi-channel EEG in terms of neuro-spike time intervals? Any resemblance for encoding neuro spikes of brain waveforms measured via EEG?
Hi Turgay, I think there is no way to assume a such likeness between EEG waves and neuro-spike time intervals. In fact, both contain encoded information! but the way to decode in each case is different.Following
- Refik Kanjhan added an answer:5Should I perform ACSF with MgSO4 or with MgCl2?
Several authors working on spinal cord acute whole-mount prep (or acute slice prep) add c.a. 1.5 mM MgSO4 to their Standard ACSF . The use of 1.0-1.5 mM MgCl2 is instead mostly widespread amongst authors working on other parts of the CNS (and what I tipically used so far in cortex, hippocampus cerebellum, brainstem...).
Given that in all other ingredients recipes are equal, is there a particular reason to add MgSO4 to spinal cord-ACSF rather than MgCl2... or is this just (as I guess) a sort of "founder effect" applied to ACSF recipes?
Apart for Magnesium (see above), with Standard ACSF I mean c.a. 125 NaCl, 2.5 KCl, 2 CaCl2, 1.2 Na2HPO4, 10 Glucose 26 NaHCO3, c.a. 300 mOsm, pH7.4 with CO2/O2 (small variations according to each author).
Thanks in advance for your Kind suggestions and contributions!
I regularly cut spinal and brainstem sections with vibratome for electrophysiology. I have tried both and have not noticed any significant difference. I guess 2 mM difference in Cl- concentration will not make a significant difference. I personally prefer MgCl2.
- Gila Behzadi added an answer:5How to identify cortical layer boundaries in live brain slice?
I am struggling to find cortical layer boundaries in mouse brain slice under differential interference contrast microscope. I typically use 400 um thick brain slice for my electrophysiology recording. Under 40x lens I kind of can see that the upper layers have smaller soma size and the soma is dense and in layer 5 the soma is bigger and sparse compared to upper layers.
The best image I can find online is this one in somatosensory cortex (http://jn.physiology.org/content/92/4/2185), in which mouse barrels in layer 4 can be easily identified.
Could anyone give me some links of good examples for identifying cortical boundaries in live brain slice?
Thank you in advance!
According to Paxinos & Watson atlas:
Barrel cortex: 2.3 mm posterior to Bregma,Lat 4 mm, V 1.5-1.8 from cortical surface (layer 4)
Motor cortex: 1.7-2 mm rostral to Bregma, Lat 1.5-1.8 mm, V 1.7 from cortical surface (layer 5)Following
- Jonny Saunders added an answer:8Patch primary human coronary artery smooth muscle cell (HCASMC) - whole cell configuration (electrophysiology)
I am trying to patch primary HCASMC, it was working fine but all of the sudden everything upside down. I am wondering if anyone have any tricks / experience on patching HCASMC.
Here is the details of condition: The cell was grown in recommended medium (contains 5% FBS) I usually plate the cell on coverslip 12h ahead of patching. The glass coverslip was treated with 100 uM NaOH and wash thoroughly then autoclave. I do not have a problem on getting giga ohm seal. I am using pipette around 2.5 to 4 mOhm.
But recently I'm having trouble getting into the whole cell configuration, >50% of the time as soon as it breaks in it's lost the cell. Even when I got the whole cell configuration, I lost it within 10 min or as soon as I start to prefuse my compound, it lost the cell. There was one weird case is the cell get to whole cell configuration automatically after it stays on giga ohm seal for couple mins.
Besides, when I looked at the microscope, at the beginning, the pipette touch the middle of the cell then, as the time goes, the pipette moves/ the cell moves to the edge of the cell. My colleagues checked and told me she did not see the pipette moves for 30 min in bath solution so I guess the pipette holder is fine.
Any advice and suggestion are appreciated and Thank you very much in advance for any suggestions
How is your perfusion set up? If the flow is too high it could cause instability as well - this is especially important since you are doing a dose-response, rate of flow can affect things like binding constants.Following
- Stephen Wilson added an answer:6Electrophysiology Recording in the Retrosplenial Cortex (RSC); is it normal to have short cell survival time compared with other brain regions?
I have been trying to patch in the RSC of slices taken from two month old mice. i have been noticing that the cells don't survive for very long after the preparation compared with other brain regions such as the barrel cortex. Has anyone else experienced something similar? Do you have any recommendations on preserving the neurons in this area for longer?
We were removing the skull by cutting around the left and right hand side of the base of the skull from the back before peeling it up and removing the brain with a small spatula. We did this to try and avoid cutting near the RSC.
Thank you for your responses! They would certainly have been useful but I have now finished the data collection part of my project so I can't go back and change anything now.
Thanks both for your help,
- Jeffrey G Edwards added an answer:4Do whole cell and field potential electrophysiology typically produce different findings?
I have produced results contrary to previously published literature in a similar experiment. There are technical differences between these studies, one of which is field potential recordings versus whole cell recordings. Does anyone know if this technical difference is enough to explain the differences in results?
If slice health is not good that could also cause you not to see plasticity (LTD) in your preparation. Otherwise, in whole cell a washout effect can occur, but is less likely if they are still seeing an effect.Following
- Andrea Moreno added an answer:3Has anyone separated (cut) the hippocampus in vivo whilst recording LFPs?
Does anyone have experience in cutting the hippocampus (to separate dorsal from ventral hippocampus) in vivo (anesthetized rat) whilst stimulating and/or recording electrophysiology? If so, would you recommend me which coordinates and methods are less damaging? (scalpel, blade...?)
Thank you so much in advance!
Thank you for the answers! and sorry for the delay in replying.
Konstantin, I contacted Victoria Talko as you suggested, but unfortunately they currently don't have those experiments running. But thank you very much for the contact!
Robert, Certainly you're right! I had found this paper from the Mosers lab in 2002 (http://www.ncbi.nlm.nih.gov/pubmed/11867718) and this is the one I'm trying to replicate, but Im not sure if anyone has repeated this procedure afterwards using electrophysiology recordings (I guess the trickiest thing for me is to keep the ephys baseline stable when you cut through the cortex).
- Benjamin Allitt added an answer:6Is there any model that tries to explain the short-term synaptic plasticity using only the spike trains?
Lots of works is done using the intracellular recordings, but I'm more interested in doing the same using only the spike trains.
So everyone is linking you to papers. Do vector strength and entrainment analyses. You'll find your answer there. Short term calcium binding may give you a physiological explanation outside of electrophysiological responses.Following
- Toshio Murase added an answer:1Does anyone know how I can get precise data about ion pumps in cell membrane?
I am searching for data about ion pumps in cell membrane to improve my model on my e-cell. My problem is lacking data of numbers of ion pumps(for instance, how many Na+ pumps in one cell) and their coefficient of ion diffusion. Would you please provide me some resources about these data，or just telling me the reference or database to search on?
I have no idea.Following
- 7where is FV, and PS?
I work on LTP recording,
A simple test with paired-pulse stimulation at ISI 40 ms should reveal what actually your recordings represent. If the slower negative deflection becomes bigger, then your recordings represent a mixture of FV and post-synaptic response (the slower tail). If the response remains unchanged or becomes smaller, then you are dealing with the fiber valley only. Or perhaps you are recording the population discharge of pyramidal neurons in response to their direct stimulation (this can happen if the stimulus is too strong and slices are bad). The positive deflection of your traces indicates that the current sink is located far from the tip of your recording electrode, hence mis-location of the recording electrode. Try to keep your electrodes far apart, use weaker stimuli and place the electrodes further down from the s.pyr.
Hope this helps,
- Jonathan David Lippiat added an answer:8How can I measure fine changes in cell capacitance?
I need to measure fine changes in cell capacitance. Does anybody know how to set pClamp 10 and axopatch 200b to have the lock-in amplifier function? I know it is possible on Heka amplifiers through the Pulse function.
It is possible to do this without lock-in. See this paper, but I am afraid that I cannot offer any guidance on the contents or implementation. I have EPC10s in my lab so never needed to go into its detail.Following
- Ruta Vosyliute added an answer:3How to measure Monophasic action potential?
In Langendorff apparatus (controlled or constant flow), what devices are necessary to acquire monophasic ventricular action potential in rat heart?
I'm also working on Langendorff-perfused hearts (mostly rabbits) using optical mapping and standard glass microelectrodes. I'm attaching a paper, maybe you will find some info there. If you need more information, let me know. Best regards!
- Norbert Weiss added an answer:3How do I dissolve heptanol into my physiological solution?
Hello, I am trying to perform gap-junction blocking experiments with Heptanol, which is not miscible with water. I have a 1M solution of Heptanol dissolved in 100% ethanol that I am adding to my perfusate to yield a (heptanol) concentration of 1.5mM. However, I am concerned that this is yielding the same result as simply adding raw heptanol to my perfusate (i.e. that the heptanol separates into its own phase). I should say that I have seen the method I am currently using in papers trying to achieve the same gap junction blockade.
Am I doing this right? If someone with experience of using heptanol for electrophysiology or calcium imaging could help me out, that would be much appreciated.
The only think you should check is that your 0.15% final ethanol has not effect by itself on your preparation (but it should be fine).Following
- Michael C Wiest added an answer:4Has anybody used Utah Array in their research? Is it a good option for rodent subjects?
I am contemplating about using them on mice
Utah arrays are about ten times more expensive than microwire arrays, so that's another reason you might want microwire arrays for rodents.Following
- Mao Yeh added an answer:9Why 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+.
Is this your situation? Every time you change your pipette, the holding current is more than 2nA and gradually decrease.Following
- Sergio Martínez-Bellver added an answer:2How do I calculate theta index?
I want to analysis of theta wave of Hippocampal place cell. Any one explain or Share some good reference about " how to calculate theta index"?
I am not sure about what kind of Index you want to calculate. However, in one of my papers I used some index in order to evaluate the relation between single units and the hippocampal theta oscillation.
Hope it helps.Following
- Tomoki Kazawa added an answer:4Which cesium-based intracellular medium to use for calcium current measurements?
I want to do some calcium current measurements in dissociated hippocampal cultures, to make an I-V plot. I suspect the mutated protein I express, influences the VGCC-conductance. In order to isolate the calcium currents I plan on blocking the sodium-currents with TTX and use a cesium based intracellular to block the potassium currents.
My problem is choosing the cesium based intracellular medium. After doing a quick the literature I have seen several mixtures. I have seen recipes with either: Cs-glutamate, Cs-sulfate or Cs-methanesulfonate. Can someone help me by explaining these compounds?
Furthermore, I have seen some use barium instead of calcium to assess the conductance of the VGCC. Why use another ion?
Thanks in advance,
When not using Cl ion, the experimenter wants to care about Cl concentration or cl activated processes( Cl channel Cl activated Ca channel and etc.).
As a replacement of cl, probably methanesulfonate is newest (improvement from sulfate) among candidates you shown.Following
- Marco Mainardi added an answer:6I'm moving to record LFP in awake rats, but I have problem to figure out how to build the chronic implant. Do you have some suggestions for me?
I want o record the LFP in different place, but I don't have a clear Idea how to do it, can you suggest me some protocol or paper about this subject?
Daniel has already provided you several essential infos... If I may add:
- you can also consider using stainless steel wire electrodes (Advent Research Materials # FE632211) instead of screws, although the latter can provide additional stability (which is desirable in rats);
- if you plan to detect "subtle" events, such as inter-area LFP differences, performing bipolar recording can increase spatial specificity;
- the whole implant should be secured in place by using, e.g., dental acrylic cement (Paladur);
- you can check PMID 24478697, 22577757, 21966482 papers I published with experiments on mice.
Hope this helps,
- 4When graphing IO curves from field recordings, how do I convert my stimulation parameters to µA?
I see papers graph IO curves as "fEPSP (mV/ms)" on the y-axis and "Stimulation Intensity (µA)" on the x-axis. How do I convert my stimulation parameters (ex: 2V at 40 µs duration) into µA? I'm using the following kind of stimulator. Any help would be greatly appreciated!
Thijs point is a good one! You can calculate the current intensity (based on the V and R) at the output of your unit but you will not be able to find out precisely the current intensity applied to the tissue due to mentioned variables.Following
- Andrey D Ivanov added an answer:3What is the best practice to perform daily maintenance of vibratome?
I am an electrophysiologist, and I prepare rat brain slices (not fixed, but directly from live animal) as a daily routine. I know that the vibratome is supposed to vibrate only in the horizontal direction, not in the vertical direction. However, every two weeks or so, the vibration in vertical direction gets really bad, and we have to recalibrate the vibratome. I have asked several labs who use vibratome in my department, and they seem to follow quite different practices to maintain and clean the vibratome.
In our lab, after cutting the slices, we unscrew the blade holder off the vibratome head stage and clean it separately, while keeping the ice tray (our post-doc believes that taking off the ice tray would make the vertical vibration worse). Another lab I asked does the opposite, where they take off the ice tray and keep the blade holder on the head stage. Yet another lab takes off neither, and they check the vibration on a daily basis.
I am trying to get collective wisdom from ResearchGate. How do you clean and maintain the vibratome? How often do you perform vibra-check?
I regularly use a Leica vibratome for acute rat brain slices preparation.
I also prefer regular razor blades, but I use one blade per day for 2 or 3 sectionings.
Of course, vibro-check is done after every blade replacement.
I do not normally remove the head stage for cleaning, but I always check all the screws before cutting.
I clean the head stage and the blade with bi-distilled sterile water and immediately gently dry it with a paper towel.Following
- Shakil Patel added an answer:9Can someone help as I am trying to fit the activation phase of my whole cell currents using function used in the attached paper but I am struggling?
I am using the pClamp 10 software to acquire and analyse whole cell currents. I am using the attached paper and trying to fit the whole cell currents to equation 2 found on page 207 and (its page 11 of 41 on the pdf document). I am struggling to find an equation similar to it using the same hodgkin and huxley type n4j fit used in the paper (link http://1drv.ms/1gySxVo) so I think it means we have to use the custom equation option but that is very difficult to use. Please can someone help me resolve this issue. I have linked some current traces for you to fit to if that helps (the link is http://1drv.ms/1gySclo).
I did email pClamp support but they have temporarily stopped the support until September.
Any help will be greatly appreciated
Hello Guys. Thanks for your suggestions. The reason I wanted to apply the same fitting equation to Cahalan et al. was because the currents I see are the same currents as the currents he found. By applying the same function to my currents it just adds another paramater to compare. When I applied the equations I was just fitting the activation phase, I may just use the single exponential function after all.
Thanks for all your help guys.Following
- Jill Miotke added an answer:8Any advice dealing with extracellular matrix/perineuronal net in acute slice patching?
Hello all! Surely those who have worked whole-cell with acute slices can commiserate with the impotent fury of seeing pipette after pipette slain, tangled in the sticky shielding of extracellular matrix. I am also working with particularly old animals, and they seem to really pack it in densely as they age.
To be specific - even after cleaning I will approach a cell, perfectly in a position that I know will seal on the rare cells that have been given a shave by the vibrotome, using pipettes that have been pulled with a program I know to be stable and effective.... and upon releasing pressure I can get the resistance to a maximum 30-40MOhm with coaxing. Upon further movement it is clear that I am caught in extracellular matrix.
I have tried everything I can think of - traditional cleaning pipettes, clearing with positive pressure, grasping the matrix with suction and attempting to tear it/work a hole in it with the micromanipulator, pulling pipettes to a point and attempting to spear it, leaving tissues for a few extra moments on the vibrotome blade in an attempt to loosen it, and even breaking pipette tips manually with a diamond blade to make rough edges reminiscent of a broken beer bottle in an drastic attempt to rip it apart or just nuclearize the whole layer of cells. The only thing I haven't tried is any digestive enzyme for lack of funding and fear that it may render my prep excessively non-physiological, but at this point I'll try anything.
Has anyone found anything that works? Even for whole-cell my sense of sisyphus is reaching a breaking point.
First, I wanted to let you know how much I have enjoyed reading your posts. You have a great humorous writing style that captures the frustration we all have at the bench. Now I will always view micropipette placement as jousting.
I am not an electrophysiologist (I just do enough to attempt in vivo electroporation and helped map visual responses with fEPSPs), but there have been several in my mentor's lab that have tried whole cell patching on the retinorecipient neurons at the bottom of the SFGS in the goldfish tectum. They are pretty small (~ 10 um), so they finally went for a perforated patch setup that used gramacidine in the pipette (evidently nystatin didn't work well). So I was following this thread to see what others had found that might work in our lab.
My question is for you and Elise. Since mammalian perineuronal net is usually rich in CSPG, has anyone tried chondroitinase ABC to clear out the ECM? I didn't have time to do an exhaustive Pub Med search, but no one seems to have mentioned using it. Unfortunately, CSPG is involved in modulating neuroplasticity, so I am not sure if it would be a good idea. But then, all the other enzymes have effects, too. Just curious.
Jonny, I am glad something finally worked!
- Joseph Valentino Raimondo added an answer:23Can 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 Kim, we still haven't bought the slicer yet so I'd be interested in how you find the EasiSlicer, and how much z-axis movement you observe. I'm still leaning toward the Compresstome but not sure...Following
- Arjun A Bhaskaran added an answer:4Which is the best way to avoid an inflammation?
Hi, I am doing Patch clamp, in vivo. Many times I am encountering an inflammation at the site of patch after a day or two!! Can anyone help me with a good solution to avoid inflammation? As I am going to try a virus, I need to keep the animal for atleast six days!
Thanks in advance
Thank you very much for the kind reply. I am doing my experiment in layer 2/3 neocortex. After the experiment I used to cover it with silicon. Still you can see an entire damage from Superficial cranitomy till layer 4-5. I felt it is because I use many pipettes, and then I reduced it into even single pipette, and again there was inflammation!Following
- Joseph Valentino Raimondo added an answer:7Which USB camera for IR-DIC?
I want to do e-phys recordings on mouse brain slices. Next to field-potential recordings and patch-clamp experiments (with IR-DIC) I also want to record an intrinsic optical signal. This should be recorded with a circular buffer that is running and updating during the experiment, so I don't miss the start of the event I want to record.
Therefore I am currently searching vor a USB camera that is compatible with the µManager softwarer.
I am not to familiar with camera qualities or technical features, but if you could tell me why the camera you suggest is the best, I'd be glad to look everything up I don't get.
Thanks a lot!
These are awesome for the price - I think the code is open too but not sure how much work it would take to get it working for your application.
- Celestino Sardu added an answer:48Young 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.
ok guys, it may be an important idea, but now it is the moment to start research together. Let me know please when we could start, I really appreciate this. regards,
- Sayantani Sikder added an answer:12What kind of anode to use for electroplating of silver electrodes?I was coating silver electrodes with bleach for patch clamp recordings. I was told that electroplating gives better and long lasting coating. I found protocols for making the electrode positive in a concentrated KCl bath, but I couldn't find any information about what the anode should be made of. Classic copper cable doesn't seems to be right.
We are using a fresh silver wire for electroplating. I couldn't find any information about the duration of the reverse polarity as well as the reason behind reversing the polarity. Moreover if we are using a silver wire for anode , will reversing the polarity not destroy the already coated wire?Following
- Manuel Castellano-Muñoz added an answer:5Has anyone measured the response of calcium indicator dyes to single neuronal spikes?
I've been scouring PubMed for a figure showing spiking in an electrophysiological trace recorded simultaneously with optical imaging of a calcium indicator dye (specifically fura and fluo-4) to no avail. The closest I've seen is a response to a single electrical field stimulus in Akerboom's GCaMP5 paper. I've also seen simultaneous electrophysiology and calcium dyes in non-spiking cells. Do we know what the single-spike response of these dyes looks like?
It is not exactly what you are looking for, but maybe be interesting for you:
Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ. Fisher JA, Barchi JR, Welle CG, Kim GH, Kosterin P, Obaid AL, Yodh AG, Contreras D, Salzberg BM. J Neurophysiol. 2008 Mar;99(3):1545-53.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.