Science topic

Electrophysiology - Science topic

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.
Questions related to Electrophysiology
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I perform positive pressure when I put my pipette in the bath, I go to the cell of interest and I manage to get to its membrane. However, when I remove the positive pressure, it seems that it does not have the necessary pressure for the formation of the seal, so I try to gently pull it with my mouth and in the same way I can't increase the resistance. I am using borosilicate pipettes with 2 - 4 Mohms, internal solution with 270 mOsm and external with 315 mOsm. I notice that my system, when fitting the pipette and adjusting the holder to the pressure system, it becomes extremely resistant, both negative and positive pressure. Negative pressure with the mouth is extremely difficult to perform and it looks like a vacuum in the pressure system line.
Thank you for helping me!
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Max gave good advice.
About pressure, different people control the pressure different ways. Some people use a syringe with plunger to apply pressure and monitor it quantitatively using a manometer. We just use our mouths on an open 1 ml syringe, and get a feel for what is a good pressure.
Positive pressure (blowing) should be less than Dizzy Gillespie, maybe more like whistling, enough to open up the tissue, but not enough to blow the cell away. Negative pressure (sucking) should be applied almost immediately after the positive pressure is stopped, and ideally is only needed for 1 or 2 seconds. The negative pressure is weaker than sipping through a straw, and nowhere near milkshake strength. I personally stop the negative pressure when the seal gets above 100 M-Ohms. It should form a gigaseal shortly after that. You should fight for a good seal, but you shouldn't have to, if you see what I mean.
Other thoughts:
* I would recommend that you patch near physiological temperature. Membranes are much more fluid and seals form more quickly. It also has the benefit of physiological relevance.
* Watch for saliva in the tubing. Sorry to be gross, but if there is any, you can't control the pressure. If need be, put a bit of kimwipe in the syringe to absorb it.
* Make sure you are patching healthy cells. Unhealthy cells are hard to seal.
Patching is fussy. But keep trying.
-Matthew
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Hello Everyone
I want to know the experience of other people and whether they have recovered at least 80-90% of the original GABA current prior to PTX application or not. Based on literature, PTX is a kind of an "irreversible" and an open channel blocker, especially to WT GABAARs.
If you have recovered 80-90% of the original GABA current, how long is your wash time, and what is your working PTX concentration in your electrophysiology experiment?
Hoping for an informative answer, advice, and recommendation.
Thank you very much.
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From personal experience, if you have the option to wait 45-60 min in wash-out, you should see the recovery (partial).
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I will really appreciate it if somebody could share the software. My lab owns the stimulator but when we acquired it we didn't get the software nor the drivers. Grass doesn't exist anymore and "Natus" which is the company that bought it told me that "S88X" is a discontinued product and they do not longer provide support for it.
Its crucial for us to control the device from a PC.
Thanks in advance
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Hi David Martínez-Vargas Janelle M Fine Eric Kenji Lee ! Do any of you still have the Grass S88X Stimulator driver and software? If yes, can you please send it to me at apoorvar@umich.edu? Many thanks!!
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We have been using HEKA Patchmaster for electrophysiological recording for several years. And for analyzing data by using Clampfit, we first exported data collected by Patchmaster into PULSE v8.6-format, and then used the ABF utility to convert the file to an ABF-format file. However, after upgrading our computer system and the Patchmaster software last week, we cannot export data as PULSE v8.6-format anymore. The export formats in the new version are ASCII, Igor Pro, MatLab, WMF, and Printer. How can we import these files (especially the ASCII format) and import them into Clampfit for analysis?
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Please find attached an ABF file which I converted from *.asc.
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Hi everyone,
I recorded the LFP signal in two different conditions of the rat brain Ca1. Under different conditions, the power spectral density(PSD) values in the delta, theta, beta and gamma frequency bands have changed. What does it mean to change the values of different frequency bands in the rat Ca1? Does anyone know the meanings of the different frequency bands of the rat brain Ca1? Or in these cases, introduce references to me.
I will be thankful for any help.
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Hi
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Hello,
As state in the title, are there any TTL pulse generators recommend for doing both electrophysiological recording and video tracking?
Thanks.
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I used spike2 from Cambridge Electronic Design (CED) for that with their power1401 DAQ and you can do it almost out of the box. I have seen it done with National Instruments equipment too, it's more flexible but takes a bit more code for implementation.
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I am recording sEPSC(holding at -70 mV) and sIPSC(holding at 0) these days. My internal solution is Cs-based low Cl solution. But EPSC still appears when I was recording sIPSC which is obviously incorrect. My collegue is doing the same experiment and using the same solution as mine but recording in his rig doesn't have such issue. Does anyone have any idea about this? I don't know which part could be blamed. I am sure the cells are good. I would be appreciate for any suggestion. Thanks!
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Dear Jiachen,
I recommend that you use pharmacological blockers to isolate your currents of interest (CNQX, APV, picrotoxin). It has the advantage that you can also check whether or not your recorded currents derive from specific ion channels, and it may eliminate your EPSCs.
Good luck!
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I reviewed many articles about the neurodegeneration of the human brain. Many researchers pointed out that complexity is a key feature in detecting alterations in the brain. However, the characteristic changes that occurred in the electrophysiological signals may depend on different factors that are shared by the subjects in the same group ( patient or control). Researchers apply some statistical tests to show obtained results are significant but I'm not sure whether the results are really related to the proposed research hypothesis ( Complexity changes with disease). We do not know that the subjects in one group drink coffee regularly while other group members do not. There are many possibilities like this for the people who participated in these experiments.
Now, this is my question;
What methodology can be utilized to ensure our hypothesis is REALLY true or not true at the end of the study? Do you have any suggestions to overcome this specific problem?
Thanks in advance.
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You touched a very tough subject that is penetrating the whole biology and medicine: the hypothesis creation and testing. Simply said, a combination of Complex Systems measures and AI together with machine is a very useful tool that can help to address similar problems to yours.
When you are really interested about complexity measures, which are employing entropy, their application to ECG (EEG is not much different), and subsequent creating and testing of Hypotheses using AI and ML, you can read the methodical paper on prediction of heart arrhythmia. The paper explains all from the first principles and have a rich list of related literature.
After reading it, when you find it useful, it would be possible to continue in our discussion, as the subject is really uneasy to grasp without thinking through it all.
Just one remark, any habits, comorbidities, and other health related features can be easily incorporated into AI & ML methods. I recommend reading papers on gut microbiota health/dysbiosis, which I shared in the project "Complexity in Medicine…" The research is linking gut microbiota dysbiosis to neurodegenerative diseases!
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The setup of electrophysiology is recording very small action potentials spikes in ALL neurons measured. The sodium currents observed are also small.The salts used was renewed and everything remains same AP. Any suggestion?
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Dear Julieta,
There can be a number of reason why the APs may be small.
What neurons are you recording from? And what is the amplitude of the AP and the peak potential?
A reason why AP become smaller (and broader) is that the series resistance (R between amplifier and the neuron) with your pipette capacitance functions as a low-pass filter. This reduces the peak and broadens your AP. Do you measure these parameters? If yes, what are the values and do you use the bridge balance and capacitance compensation to minimize the effects?
For more information, Barbour’s Electronics for Electrophysiologists is good place to start (https://www.biologie.ens.fr/~barbour/electronics_for_electrophysiologists.pdf).
Good luck!
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Dear freely-moving e-phys-community,
in my PhD project, I am faced with the challenge to target the posterior insula, which is located in the ventral half of the brain, in freely-moving electrophysiology in rats.
Before implanting the silicone probe, I level the skull by adjusting
(1) bregma and lambda at the same level on the dorsoventral-axis for anterior-posterior-levelling and
(2) the parietal bones +3.5 mm and -3.5 mm lateral from the interfrontal suture (approx. in the middle between begma and lambda) at the same level on the dorsoventral axis for mediolateral-levelling,
with the stereotaxis device from Stoelting.
After dissection and slicing, I find the trace of the probe going more or less tilted in both, the AP and the ML-axes. Since I am targeting a quite narrow spot in the far-posterior insula (AP -2.9 mm, ML 6 mm, DV 6.2-8 mm from bregma, according to the Paxinos & Watson atlas, 7th edition), I miss the spot because of the probe going diaginally. The mismatch is different across trials, probably due to individual differences (asymmetries) between animals.
Does anyone have an advice for proper skull levelling, or any other solution to make the probe travel staight vertically, for targeting deep (ventral) brain areas?
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Perhaps you can mount or glue a stiff tungsten or stainless insect pin onto the side of the the silicone probe to make it stiff enough to withstand any lateral or medial torsion resulting from differential densities of different dorsal brain regions that you need to penetrate to reach the insula. I think your coordinates are good but if you make sure all stereotaxic arms and attachment of your probes are completely tight and use another probe to make sure the skull is precisely level between lambda and bregma your results will probably be much more accurate.... Good luck. Dr. E.K. Walls
you can send me a photo of your probe and I can give you advice better if I see it ok. email: wallse@purdue.edu
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Hi everyone,
We recently bought a new amplifier "Axoclamp 900A", for the Intracellular recording setup, previously we were using "axoclamp 2B". We also bought Pclamp11 software to work with the new amplifier. We use "Digidata 1440A", a digitizer in our electrophysiology rig. So, I have connected the I-clamp command(channel 1) and V-clamp command to the analog output ports of the digitizer. and the scaled output(channel 1) to the analog input port to the digitizer, along with this I have installed the Pclamp 11 software on the computer, for setting up the protocols for intracellular recordings, for example, AHP, FI, etc I did it with the episodic stimulation and putting parameters in the waveform.
The problem which I am facing is during the experiment we need to see the step on the monitor and as we move the electrode slowly in the slice we are able to detect small spikes and eventually the bigger once(due to which we know we are in the cell and now we can perform different protocols in our experiment. I am not able to detect any activity when I move the electrode into the slice, basically no signal. until we get that we can't do anything. I have set the gain to 100 in axocommander and the highpass filter to 300hz and lowpass on bypass mode.
We previously used Master-8 attached in our set up for stimulating the cells.
Using the model cell I am able to get an I-V curve.
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Hi there!
I hope that you were able to solve your problem.
I am contacting because I would like to know more about your with Axoclamp2B, if possible.
Our Digidata 1322A was connected with pClamp8.2 but it stopped working (DAC0 error). So we might need to start using AxoClamp2B. Could you briefly describe how you managed to make it work?
Thanks!
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I am trying to follow this paper https://www.cell.com/biophysj/fulltext/S0006-3495(02)75543-4 for finding out the stoichiometry of one of the exchanger in question. However, even if I use a massive gradient of the ions involved I do not get a shift in reversal potential as these authors have got in this paper. I am using the holding potential of 0mV and then applying a ramp of -100 to +100 mV. I am curious if the holding potential is playing a role in dictating the reversal potential.
Any leads would be highly helpful. Thanks!
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Dear Sourajit,
In the paper, another characteristic is that the -100mV initial hyperpolarization brings the Vm to around -20 pA, while yours, it goes to more than -500pA. Are you performing any leak subtraction? If not, I advise you to, otherwise the leak current could be too big and just mask all your results. In the paper they do it pharmacologically using LiCl and a post hoc subtraction:
"The currents observed due to voltage ramps in LiCl/EGTA bath solution (conditions preventing the exchanger from operating in either direction) at the start of the experiment were digitally subtracted from subsequent currents obtained under various conditions, and the resulting I-V traces are shown in Fig. 3 B"
Btw, using conventional p/n leak subtraction will be ineffective if your exchanger isn't voltage dependent.
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There's a weird issue. I have a stim box, which has 2 entrances, one called "act.", and another one - "ref". So if I plug a bipolar electrode in it, so 1 electrode goes to "act." and another to "ref", I see bubbling on only one tip, instead of seeing it on both. If I exchange places, where electrodes connect, I see bubbling on another tip. As I know, it should not be so.
So, do you know, what's the problem here?
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Great! Happy to help.
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I need training in rodent's brain electrophysiology.
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Then probably your best options are either to attend a school/workshop that also teaches ephys (but you would gain only limited experience), or to look for a postdoc in which you can combine both ephys and also the techniques in which you are already trained. Good luck! :)
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Electrophysiological signals are generally measured using Ag/AgCl electrodes due to their non-polarizing properties.
Polarized electrodes are unfavorable for electrical stimulation and potential measurement.
Have you ever paid attention to the polarization of nanocarbons and carbides?
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There are two main types of electrodes: (1) glass micropipettes filled with an electrolyte solution (2 or 3 M sodium chloride or potassium chloride); and (2) metal electrodes (usually tungsten, steel, or platinum–iridium). An important characteristic of both kinds of electrodes is their electrical resistance, which is related to the exposed tip size. Smaller tips have higher resistances, and they restrict the area from which potentials can be recorded, thus permitting the isolation of the activity of either a fiber or a cell. Large tips and low resistances pick up the activity from a number of neurons and are of limited use in efforts to identify the functional properties of single cells.
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Hello
After transfecting my HEK293 cells with a construct containing an opsin and fluorescent reporter protein (e.g. ChR2-mCherry), I tried to patch one of my transfected cells based on red fluorescence of mCherry for electrophysiology and optogenetic experiments. Then, after patching and stimulating light, I notice that there is no current from this cell even though it produced red fluorescence under the microscope before patching
Have you experienced this kind of phenomenon?
Thanks.
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I have not experienced this in my work
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Hi everyone,
Is the LFP frequency bands different for rat and human brains?
What are the LFP frequency bands for rats?
I will be thankful for any help.
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Dear
Megan Sholomiski
, thank you for your answer.
I find the following result when I search the frequency band in Google.
" LFP frequency bands: The classification of the LFP into distinct frequency bands, known as delta (approx. 0-4 Hz), theta (4-10), alpha (8-12 Hz), beta (15-30 Hz) and gamma (30-90 Hz) has been adopted from the EEG literature. It is based on the strong correlation of each band with a distinct behavioral state. "
Is this classification also valid for rats?
Why do frequencies 8 to 10 belong to the two classes and why do frequencies from 12 Hz to 15 Hz not belong to any class? I also found different results in the articles. Anyway, what is the valid classification for frequency bands for mice?
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Hi all,
There are obstacles that I have faced during electrophysiology recording for the neurons derived from stem cells. Neurons were fell out once I started patching cells. I cannot record any actional potential and/ or sodium or potassium current. Although I have good neurons in my culture. I would appreciate it if you can give me advice to overcome this issue. Thank you.
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Hi Randah. Cultured neurons can be a little finicky to work with. From what I understood, you can't get anything from them. So let me ask: how is the seal (leak current)? And the resting membrane potential in current clamp? Did this preparation work smoothly before? What's the Na concentration in your culture media?
Andre
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SNAP
How to use SNAP as a nitric oxide donor correctly? Is it possible to prepare a stock solution and store it in a freezer, and prepare the working concentration by dilution before application?
Or should SNAP crystals be dissolved immediately before use?
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The reagent SNAP, are prepared by mixing solutions of N-acetyl-penicillamine, with equimolar NaNO2 in 0.5 N HCl. The reaction is typically complete within 1 min; for reasons of stability, the solution is neutralized by addition of NaOH immediately before addition to the protein solution. source: Nitric Oxide Synthase: Characterization and Functional Analysis
Ying-Yi Zhang, Joseph Loscalzo,
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Hi all,
I am conducting 10 minute gap free voltage clamp recordings in hippocampal slice cultures, and would like to be able to monitor the Cm, Rm and Ra at regular intervals (possibly every two minutes) (i am using clampex 11.1). Is it possible to periodically integrate an episodic stimulation event into a gap free recording, so that i can look at and calculate these values from the capacitive transients?
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You may apply a linear ramp pulse any time to evaluate any change in access resistance.
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I have doubts about which is the best and most suitable amplifier. We own the AxoClamp 2B and the Axopatch 200B. Thanks a lot for the help.
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Depends on your budget. If that's not a problem, get yourself an Axopatch 700B. You can do more than just field potentials. You can, when needed, do whole-cell experiments (say, when buffering internal Calcium with EGTA or Bapta, blocking a channel from the cytosolic side...etc.,) and unitary activity channel recordings (all configurations: C-A, I-O or O-O). Furthermore, you can perform , ion-selective electrode recording, amperometry/voltammetry and bilayer recordings.
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Hi
I'm new to electrophysiology and have been reading some papers. But i can't really understand how to read their results. For example, attached here is a screenshot from a paper (Hypoxia reduces mature hERG channels through calpain up-regulation Shawn M. Lamothe,* WonJu Song,* Jun Guo,* Wentao Li,* Tonghua Yang,* Adrian Baranchuk,† Charles H. Graham,* and Shetuan Zhang*,1)
For the patch-clamp recording, why are there so many curves? are those repeated measurements of the same sample?
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The concept of current-voltage (I-V) relationship is one first thing that any electrophysiologist learns. Refik explained the data nicely. the authors of the paper you showed did more than most electrophysiologists do which is to draw the exact voltage protocol they used (see inset in B above hypoxic conditions at 24h traces) to generate the family of corresponding recorded currents. Graph B also has the scale bars to give you the exact time of how long they held their membrane voltage to any of the voltages tested. I did not read the paper, but for some reason the I-V relationship was not generated from the recorded traces nor the inactivation-Voltage relationship. Those measurement, if generated, would give more info on how hypoxic conditions affect the channel's characteristics. All I see is a time-course (TC) of the hypoxic condition (which, of course, is important) with time after 6, 12, 18 and 24 hours. I guess, all I want to say is that for a beginner in Ephys., you don't need such a protocol to generate that time-course. You could do the same just by depolarizing the membrane to one voltage followed by another one to see the tail current. To learn more, I recommend that you read two essential books: The first one by the late Areles Molleman - Patch Clamping, An introductory Guide to Patch Clamp Electrophysiology and of course, the second book (the Ephys. Bible) by Bertil Hille - Ion Channels Excitable Membranes. The first book is free to download from the web. Happy reading :)
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I am starting to use BiC/4AP for an experiment to stimulate hippocampal neurons. This technique has been used previously by other labs and a former member from my own lab. I have tried several times, but cannot seem to get the same results as others. I am using bicuculline and 4AP from at least 10 years ago that has been stored at room temperature in a dessicant box. The bicuculline is stored in aluminum foil also to prevent light exposure.
I'm wondering if my experiment is not working because the drugs are too old. I have tried to look for the shelf life of the drugs but cannot find much. Does anybody have experience working with these drugs and have any idea of how long they are good for when stored at room temperature?
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Long term storage of bicuculline should be at -20 oC and this will only be useable for approximately 1-2 years. 4-AP should also be stored at -20 oC for long term and will only be useable for approximately 6-12 months.
Therefore, you should discard the old solutions and order fresh products.
(An tip for identifying shelf life is to look at the stability and storage section in the product information sheet for each product)
Hope this helps!
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Hi all,
I am trying to compare the conductance analysis from a few channel currents that I have analysed and I am trying to understand the effect of mutations I am studying on.
I always fit my conductance data with the Boltzmann exponential, from which I use the V50 and slope factor values to determine the voltage dependence properties of the current and hence be able to compare my data. Whilst for me, V50 comparisons are super easy and understandable, I always end up confusing myself with slope factor comparisons. I try to think of it as "if the slope is high (the shallow curve) there is lower voltage sensitivity as with a given change in membrane potential, there is very little change in conductance" and "if the slope is low (curve is steeper) there is higher voltage sensitivity as with any given change in membrane potential, there is larger change in conductance".
Does that make sense? or am I confusing myself yet again.
Thanks everyone for your advice.
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It is said that the Fermi-Dirac distribution would be
better to be used
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Hello. I'm looking for a cell line stable expressing CaV3.2 (CACNA1H, T-Type) ionic channels. It will be for eletrcophysiological studies (manual and automated patch-clamp).
Thank you in advance!
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You can get HEK293 Cav3.2, see Cellosaurus entry:
As shown in the entry it is available from Kerafast.
Best
Amos
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Recently Jamali et al., at Ziv Williams' lab in Harvard published an intriguing paper in Nature:
regarding the cellular basis of theory of mind which I believe is one of the first hand evidence to prove mentalization at cell and circuit level. The methodology was based on single cell electrophysiology which seems interesting yet tricky as it may spark this philosophical dilemma of systems neuroscience that complex behaviors such as theory of mind may be originated from synchronized population activity in downstream path that may not be represented or evoked in individual neuronal activity in dmPFC. However, Jamali et al. could rationally and intriguingly conclude and investigate such a complex behavior at a single cell level. Interestingly, they indicated that dmPFC neurons can predict whether contents of one's beliefs in the big picture would be true or false.
  • While study of theory of mind at cellular level seemed almost impossible before this, and studies shifted also to cellular and circuit level with this landmark publication, what would you think should be the future research on theory of mind? What is the big question and hypothesis if we want to use multi-modal neuroimaging approaches?
  • What methodology and approaches would better decipher impaired theory of mind in psychiatric diseases such as schizophrenia and autism spectrum disorder?
These are just a couple of questions that may emerge but feel free to discuss and contribute to this topic from any aspects that you would think would give us a better view of the underlying mechanisms of theory of mind.
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Bahman Sadeghi thank you for sharing this article, the methodology is so thoroughly described that it made it possible for me (with my limited knowledge of brain research methods) to get to a fair appreciation of the extent to which the study is supporting theory of mind. It also made me think of the value of the theory more broadly, so much so that I decided to draw from it for my own research !
Gabriele Scheler with the risk of sounding ignorant in assuming I can make any contribution to the topic given that I have a very different kind of scenario (from state-of-the-art lab research) in mind when trying to make sense of the theory, I would very much be interested in being part of the conversation :)
R. R. Poznansky I've found your answer very reassuring, the scope of accounting for unconscious processing in explaining the "mind" is precisely the reason I have decided to use theory of mind in my research, because I'm looking at a phenomenon which is poorly understood in terms of causality as a result of researchers trying to avoid the intentionality dilemma when investigating human behaviour.
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Hi there,
I've been noticing that after adding my blocking chemicals (TEA-Cl and CdCl2) to isolate sodium current, my pH increases beyond my ideal point (I'm trying to stick around 7.4 and it drifts to around 8.0 after adding both) and there is first cloudyness and then precipitate forming in the solution after addition of the CdCl2.
Prior to adding the blockers, I am bubbling my ACSF with carbogen for twenty minutes. I also do not add sodium bicarbonate or dextrose to my 10X stock. My concentrations for ACSF (working solution) are as follows:
125mM NaCl
2.5mM KCl
1.25mM NaH2PO4
1mM MgCl2
25mM NaHCO3
25mM dextrose
2mM CaCl2
75mM TEA-Cl
0.2mM CdCl2
Again, target pH is 7.4. Would really like some input on this, as well any any relevant chemistry as to what is happening. Thank you!
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Cadmium binds to phosphate and forms an insoluble compled that precipitates out of solution. If you wish to block calcium currents, leave out the NaH2PO4.
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Hi, I used A.M.P.I. ISO-Flex stimulus isolator for evoked responses in patch clamp.
Now I moved to another lab and I need to buy a new isolator.
Do you know any good isolator you like? or A.M.P.I. will be good enough?
Thank you.
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Hi
I might be a little biased, but consider looking at the Digitimer DS3 constant current isolator, which is very popular amongst brain slice electrophysiologists. It also has a unique output discharge circuit that prevents capacitance build up during repetitive stimulation.
Regards
Gareth
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Hi all,
I make my aCSF using the following: 126 mM NaCl, 2 mM CaCl2, 10 mM glucose, 2 mM MgSO4.7H2O, 3 mM KCl, 1.25 mM NaH2PO4.2H2O and 26.4 mM NaHCO3 bubbled with carbogen gas (95% O2, 5% CO2). The pH is usually 7.2-7.4.
However, recently I checked the pH of my aCSF (at 30 degrees with bubbling) and it was 7.85.
To get the pH to read 7.4 I had to reduce the concentration of NaHCO3 to 10.6mM. This reduced the osmolarity and so I added 17mM NaCl to balance it back to 300mOsm.
I have since recorded hippocampal brain slices using this new adapted aCSF and the cells look fine and I can record LTP.
Does anyone have an idea as to why the pH has increased? Or has anyone seen a lower concentration of NaHCO3 used in aCSF before?
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Pls doubecheck the data
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I compensate my series resistance 75-80%, I have sodium currents that range from 0.5 to 6 nA, and my pipettes are 1.5-3 megaohms.
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If we talk about an inward current (like sodium current in cardiomyocites), should we then put another minus in the equation?
For instance I have a desired voltage of -50 mV and a peak current of 2nA...
Then Vm= (-50mV) - (- 2nA * 2megohms of residual Rs after compensation)
Then Vm= -50- (-4mV) = -46mV
Is this correct?
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Hi everybody!
I am establishing a protocol for organotypic brain slice culture in my lab. We need it for electrophysiological study and the standard method with growing slices on membrane inserts didn't prove useful, so I opted for the one in which slices are glued to the coverslip using plasma/thrombin clot.
Nevertheless, the pipette used for electrophysiology can't go through the clot and we will need to have it dissolved before the experiment. Does anyone know safe methods to perform it and not influence the slice underneath? Logical solution would be using plasmin, but I am not sure how well it works in vitro and have no possibility to test it without buzing it first.
Thanks a lot!
Marta
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If I understood your question you need to fix the slice somehow to avoid it floating while patching, right? One way to pin the slices down in your chamber without using a metal harp is making them adhere to a smaller coverslip (usually a round 10 or 12 mm one) pretreated with poly-l lysine before transferring them to your rig. You may find these poly-L lysine-treated coverslip ready for purchasing or treat them yourself.
Hope it helps! Good luck!
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I am planning to perform in vitro electrophysiology experiments on rats model. Is it possible to store whole rat brain for invitro electrophysiology experiments or one have to perform the experiment on fresh tissue. How long such samples can be stored and at what temperature before performing experiment and what kind of reagents/solutions are required for storage.
Kindly guide.
Thank you
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I agree with Max Anstötz in general. Mammalian brain tissue requires continuous temperature control, oxygenation, nutrient supply, and waste removal if you want to study electrical activity that resembles physiological. On the other hand, there are ways to keep brain slices in culture for up to several months, through the tissue "flattens out," and the neuronal phenotypes may drift over time as some synaptic connections are lost and others change. You may want to look at the work by the Gähwiler laboratory in Switzerland.
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Xenopus laevis oocytes are used in electrophysiology to make transient expression of injected mRNA fragments. Through the technique of 2-electrodes voltage-clamp we can record the physiological phenomena that originate from the activity of the generated protein that has been embedded in the oocyte membrane. Depending on its activity we will observe hyperpolarization or repolarization of the membrane, in the presence of different external stimuli (different concentrations of anions, amino acids, organic acids, etc.).
In our case, electrophysiology recordings in Xenopus laevis were assayed. A plant's membrane transporter working in an animal cell membrane.
But, in young and old, well-fed, and apparently healthy frogs, sometimes the experiments could not be as brilliant as on other occasions. The oocytes were of poor quality, without showing that polarized two-color differentiation, and after injecting the RNA they exploded most of them. Surviving oocytes showed strange electrical behaviors, mostly related to passive endogenous chloride input channels and ended up lysing soon.
Have you had any similar experiences? If you have an explanation I would be very grateful. We are trying to "dust off" old jobs that were abandoned in a drawer.
I want to thank Dr. Omar Homero Pantoja from IBT-UNAM (Cuernavaca, Mexico) for teaching me during my stay in his electrophysiology laboratory.
#xenopus #oocytes #2EVC #electrophysiology #recordings
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Hi Franco,
I hope things have already worked on your side.
I went through the same issue from March to May. I tried to adjust all the parameters I could think of (bought new frogs, tried a couple of collagenase and concentrations, remove follicular membrane manually, different types of solution, glass vials/falcon tubes, temperature, time, autoclaved everything, etc) but nothing worked. But after we entered June, the problem just disappeared by itself by using our original protocol. I can only say that the "bad period/good period" thing is real. We sometimes just have to be patient.
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I aim to study the electrophysiological properties in pyramidal neurons in the anterior cingulate cortex (ACC) and interactions between astrocytes, interneurons, and pyramidal neurons. There is a difference between B6J and BJN in emotional behavior. I will do behavior experiments on B6N but I ask if I can do electrophysiological tests on B6J due to their availability.
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Hi Gürsel,
The differences of the genetic backgrounds are important. Also, the maturation of the neurotransmitter systems may differ with age. We have previously compared the EEGs of Bl6 and Bulb-c during basal activity as well as following an epileptic trigger.
Here is the paper:
Regards
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I am using Clampex V 10.7.0.3. I am unable to make any changes to protocols, as clicking editing option always freezes the software. Has anyone faced similar issue?
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Hi Mohammed,
As far as I remember we experienced this type of problem because our windows system was in spanish, and changing the windoes system to English solved the problem. That is at least one possible source of the problem.
Regards, Enrique
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I'm measuring fEPSP in the CA1-CA3 hippocampus region and lately I have had presence of bubbles in the bath where ACSF comes in. This leads to unstable recordings and from what I understand the bubbles are caused by the in-line solution heater. I have been thinking pre-heating the ACSF in a beaker where it's perfused before it goes to the in- line solution heater and was wondering what kind of heater you guys might be using out there. Preferably one that is not know to cause any electrical noise and small so that it fits/gets completely submerged in a 500ml beaker. Thank you!
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Hi Deogratias,
We have used by many years the Warner Instruments SHM-828 with multi inline heater and it goes very nice and very easy to use.
Regards
Enrique
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Hello,
I am almost done setting up a field recording rig. It has a transparent interface chamber and a low magnification microscope with side lamps, so I will likely need to use a dye in the recording pipette to enhance contrast and ease positioning. I was looking through the literature and it seems that Pontamine sky Blue (PBS) aka Chicago/Niagara/Atlantic/Diacotton/Amanil sky blue is often used, usually at a concentration of 2% (~20mM).
I also came across this article (https://www.jstage.jst.go.jp/article/jjphysiol/54/1/54_1_61/_article) from 2004 where they puffed PSB at similar concentration (from 1 to 3%) on several brain structures of different model organisms, and report various neuronal effects. They also cite this paper (https://onlinelibrary.wiley.com/doi/abs/10.1046/j.1471-4159.1995.65010096.x) from 1995 that reports Chicago Sky blue (CSB - apparently just another name for the same compound) having a similar structure to glutamate and being an "efficient glutamate uptake inhibitor". Throughout their experiments on synaptosomes, they used concentrations ranging from 160nM to 1mM and showed significant effects on various measurements at these concentrations. For example they determined than CSB's IC50 (concentration needed to inhibit 1/2 of the biological process (here uptake of glutamate or GABA)) was 3uM for glutamate uptake and 473uM for GABA.
It seems this compound has an important effect of neuronal functions, even at low concentrations, with various outcome depending on the brain structure. Yet, it seems it is still used routinely in electrophysiology as an inert dye. The SDS page of Chicago Sky Blue 6B on Sigma-Millipore doesn't mention any neurotoxic effect. I am wondering if I am missing something. Have these two papers been refuted since then? Is it common knowledge that we shouldn't be using them anymore? Are there inert alternatives to it?
Thanks
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You are asking the right question and exposing a little secret that everybody puts under the carpet. we (the lab) used voltage, calcium and pH dyes decades ago, all of them appeared toxic although the tissue tolerated them and even had excitation waves, my mark of good enough. you will have to find your good enough yourself. to see nice pictures look at
  • DOI:
  • 10.1016/S1350-9462(96)00038-9
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I am trying to use WINWCP instead of axoscope.
I have a Axopatch 200B amplifier and Digidata 1550B digitizer.
When I added the digidata 1550b in the lab interface option, it has been giving me this error:
DIGD1550B.DLL- DIGD1550B_CountDevices not found
DIGD1550B.DLL- DIGD1550B_FindDevices not found
DIGD1550B.DLL- DIGD1550B_GetErrorText not found
and several such windows one after another, with only the option of clicking OK.
And then it gives a final error that it winwcp needs to shut down...
Anybody face a similar issue?
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Hi Pranali,
I recommend you to contact molecular devices.
Best wishes.
Luis
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Hello fellow neuroscientists and others!
Here is probably a naive question but I am struggling to find an answer...
Are back-propagating action potentials a common phenomenon in dendrites in vivo? Or is it only an artefact of in vitro studies?
Thank you for your help,
Regards
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The question asks about action potentials, dear Bernard.
Dear Lora if you want to be a neuroscientist, learn the jargon and think before asking a question, orthodromic and antidromic propagation are jargon words. and yes it valid for synaptic transimission also.
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I read papers that report several neuronal properties between experimental and control conditions and wonder how I should be selecting data to report on. I see some trends. Spike half-width, rheobase, input resistance, capacitance, and RMP are often shown. Some include others.
Why? How do I choose which to share. Do people report statistically significant findings?
I am wondering what data to report and why? Are these data points often include to please manuscript reviewers?
Can anyone point me to any literature to explain what neuronal properties are most important to show?
I use CLAMPfit software to analyze electrophysiological data and see a lot of options and measurements taken from our readouts.
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There are a lot of standard measurements, as you’ve noted: resting membrane potential, resting input resistance, threshold potential, rheobase current, sag potential, etcetera and so forth.
For publication, all of them are equally important (or unimportant). You never go wrong by reporting everything.
But say the question is: what is important to report? Then the answer depends on what you think is going on. If your drug or genetic modification or behavioral training affects BK channels, then you’d want to concentrate on fast afterhyerpolarization and/or spike shape and broadening. If it or they (are hypothesized to) affect HCN channels, then concentrate on sag, rebound, and impedance peak. If SK channels, then medium/slow afterhyperpolarization and spike frequency adaptation. And so on …
I don’t think there is master set of measurements every physiologist should make and report. Really, it depends on what you expect or imagine in going on.
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I am recording mEPSC (at -80mV, 2micromolar TTX) and mIPSC (at -60mV, 2uM TTX, 20uM APV, 50uM DNQX) in amygdala C57 mice (8 to 10 weeks).
there is a report which says a sodium resistant channel Nav1.5 is present in amygdala, but it is usually reported to be blocked by 1uM TTX, to be safe I am using 2uM TTX...
But, my supervisor insists that if I see large amplitudes (compared to the more frequent smaller events) and very fast rise time and two decay phases, initial sharp and then slower... then it is a spike (sodium channel mediated) and not a synaptic event... both for mEPSC and mIPSC, additionally if there are these large events one after another, then its a regenerative process where sodium channels are activating themselves in feedforward loop to cause these events...
I've added a file to show which traces are rejected under this criteria.
What parameters are usually used to select mEPSC and mIPSC events ? What are the criteria used to exclude an event?
I cannot find any papers mentioning this phenomenon of TTX- R Na current in mEPSC and mIPSC events ...Please help. IS there any EXPERIMENTAL way I can prove there are no sodium channel currents in presence of 2uM TTX?
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to settle the argument, at the end of your experiment, throw in some DNQX or Gabazine to block your minis (EPSCs and IPSCs respectively) and record for 3 to 5 minutes. If you see anything left, your supervisor has a point. If you don't, he has none :)
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Hello everyone,
I am trying to record single channel events on my HEK293 cells stably expressing Cav2.2 channels, using the cell-attached patch clamp technique.
I used the following solutions:
Extracellular: 150 KCl, 10 HEPES, pH 7.3 with KOH
Pipette solution: 110 BaCl2, 10 HEPES, pH 7.4 with KOH
Unfortunatelly, I haven't been able to find the channels so far.
- I want to step the cell to 0, 10, 20 and +30 mV from a holding potential of -100 mV. I understand that to hold the membrane at -100 mV I have to set the V-command in my protocol to +100 mV (and steps to 0, -10, -20, and -30 mV). Is this correct?
- In that case, the currents I expect are patch inward currents (barium ions flowing from the pipette into the cell). I understand these currents should appear as outward currents (positive) in my recordings, is that right?
- Should I invert the polarity of both voltage and currents for analysis and representation?
- Finally, I am trying to use the Patch mode in my Axopatch 200B amplifier, but every time I try this a regular spike noise will appers. Do you know why this might be happening?
Thank you very much for your attention!
Diego
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I am also trying to get some single channel recordings of the Nav.
The solutions look fine in my opinion, but the protocol in my opinion is not correct.
The holding that you have in figure 2 is 0mV and not +100mV. If I am not mistaken in the Clampex software when you create a new protocol you decide the holding potential under the option outputs and there you put the desired holding.
Afterwards in the option waveform you can decide the first step and the delta level.
And yes the current you expect to see in the raw recording should be "positive".
About the regular spikes in the Patch mode, I was also wondering and decided to ask the Axon support and this was their reply "Instead of resistor, a feedback capacitor is used in the Patch mode in Axopatch 200B amplifier. The voltage across the feedback capacitor cannot ramp in one direction forever. At some point the capacitor voltage will approach the supply limits and the integrator must be reset to start again near zero volts. Thus the current record must be interrupted for 50 us while the integrator and differentiator reset. The frequency of resets depends on the current passing through the headstage, with the larger current requiring more frequency resets."
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I came across two papers (Shcheglovitov et.al., 2013, Nature; Zaslavsky et.al., 2019, Nat Neuro) where they mixed neurons from two genotypes and seeded them on a bed of rodent astrocyte. How does one use this setup as a readout for any phenotypes?
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Yojet,
A nice advantage of neuronal co-cultures of this type (when one can distinguish between the different cells) is that they allow analysis of the behaviour and morphology of both types of neurons side-by-side while reducing culture and staining variability to a minimum.
In the particular case of these papers, it seems to me that the authors want to analyse Shank2/3 role at the postsynapse while keeping presynaptic terminals as WT. Therefore, a "sparse" co-culture helps analysing morphological features while also provides a source for mutant postsynaptic compartments as well as WT presynaptic buttons to form hybrid synapses. I will here admit that I haven't fully read the papers and hence my view could be an oversimplification. So, this is what I got from an overview of the results and what I can assume from my own experience in co-culture.
Hope this may help you.
Cheers,
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Dear collegues,
how do you would perform Immunofluorescence staining on thick brain slices (around 400 micrometres) upon acute slices pharmacological manipulation or electrophysiology?
-cut and then stain or whole mount stain and then cut?
Do you have references (written/videos) for it?
Thank you
Viviana Triaca
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Hi there,
There is really few papers in which what I am going to say is clearly stated but:
You don't need to cut.
I have done this experiment with 350um slices
[supplementary figure 2, protocol in material and methods]
and I can tell you that if the antibodies you are using are good enough you can get high quality images without cutting. But it is really important to have good antibodies. You will probably need to adjust (increase) incubation times with antibodies, washes, and also the concentration of detergent.
Check also this paper, in which they perform IHF on organotypical slices without cutting
However, if you need to cut the slices or just want to do so, cut first and then proceed with the IHF protocol.
Hope this helps,
Cheers,
J
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I am using a Sutter electrode holder for my patch clamp experiments, but I'm having trouble keeping positive pressure inside my electrode due to a leak out of the back of the holder.
I have added small plastic tubing to the inside of the holder around my silver wire, but I am still experiencing leaks.
Any tips/suggestions on preventing these pressure leaks will be appreciated. Here is a video of me adding 3cc of pressure from a syringe: https://streamable.com/veb0cm
(Ignore the bubbles coming out of my electrode, I had performed this multiple times with this same pipette. The electrode is filled with di-water for testing purposes).
Thank you!
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Hope you have fixed this by now. There is a gasket for the back end (silicone or a red rubber like gasket which allows just the silver wire. The gold coated pin should rest over the silverwire passing through the gaske and the gasket should fit snugly.
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Dear all,
I would like to start a discussion and sharing of resources and knowledge on the use of Python for the analysis of ephys data. For the past years I have used Igor Pro and am very eager to migrate to Python as it is free, open-source and has many toolboxes available.
After a little research I found a package called Neo (http://neuralensemble.org/neo/). Does anyone know good packages to analyze voltage- or current-clamp recordings? EPSCs, mini EPSCs and such?
Furthermore, are there any packages or resources for Python that you deem worth sharing with the ephys community?
Thank you for your support
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I used neo for a long time, but now I found something that is way easier. The package is called pyabf, and you can open your file with two simple lines of code. It actually displays the entire metadata that the abf file contains, easy to use, readily converts the files to numpy arrays, and is fast. I highly recommend using this package for everyone!
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Hi everyone,
I have been trying to get a gigaseal from primary cell-culture cells from rat’s DRGs, but without success. I don’t know exactly what the problem is.. I have tried after 2 hours form getting the cells, after 24 hours, and 48 hours, I have changed the glass used to fabricate the pipettes, (I tried both thin walled glass and thick walled glass), I have tried with different pipettes resistances (from 2MΩ to 10 MΩ) but without success.
A PDF that I prepared is associated to explain what I have done, containing screenshots of what I get in the “SutterPatch” Program, and picture of the pipettes I used.
Do you have any recommendations or a solution to help forming gigaseal ?
Thank you in advance!
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Thanks very much, we will try this and see what we will get
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According to the vertebrate literature, I got the impression that head-direction cells are always anchored to an allocentric reference frame. This can easily be tested with freely moving animals. But what is about tethered animals that can freely rotate around their body axis but can`t change their actual position. Are neurons encoding the animals heading automatically defined as allocentric ones? Or how could you test with a tethered approach, if the neurons encode heading in a allocentric reference frame or in an egocentric one?
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Please correct me if I am wrong ,but the neurons (of the parietal cortex) reported in the PNAS paper are not classically circumscribed as head-direction cells. Sure, they show heading tuning but for me this tuning looks more like a sensory-based peripheral tuning. Although not being an expert in that field, I Interpret the parietal neurons to encode more general aspects of body-posture relative to surrounding objects and not necessarily to be specialized to purely encode aspects of navigation like cells of the hippocampus network. I guess this may be also the reason, why the authors do not explicitly termed the parietal cortex neurons as head-direction cells. But it is valid to say that there has to occur a transformation from egocentric to allocentric reference frames somewehere in the brain (and obviously also a retransformation from allocentric to egocentric).
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Hi all,
I am new to electrophysiology, and I have some inqueries about current-voltage relationship.
Why some authors plot both I-V relationship and I/Imax-V relationship. What is the difference between them?? And why they sometimes show some differences in statistical analysis and/or significance at some potentials when comparing the different treated groups ? For example,.there may be sigificance in I-V plot between gpA and B which disappear in I/Imax-V
2. How can I know that a drug has reached its steady-state of action for example; AICAR.
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When on plots the absolute value of I versus V will be different from one plots the normalized value of I versus V. The normalization expresses the profile of the curve such that one can compare the different cases according to the shape of their profiles. Absolute current values contains more information about the speed of the process. Which may be due to larger area of the electrodes for example.
This is a generic answer for your question. This is process of scaling is called normalization.
Best wishes
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Hello everyone,
Is it possible to calculate the E/I ratio on single-cell levels only based on miniature signals (Excitatory and inhibitory)?
If yes, then what parameters should I take to calculate it? and how?
Currently, I can get lots of parameters from mIPSPs and mEPSPs such as the number of events, decay and rise time, area, baseline, noise, half-width, 10-90 slope, etc...
Which one of these can i use to calculate the E/I balance and the calculation mathematical formula?
Thank you!
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What do you think the E/I ratio is? What do you hope to show? If you wanted to, you could calculate the total charge transfer due to mIPSCs, and the total charge transfer due to mEPSCs, and take the ratio of those, but what do you think that would demonstrate? Do you think that metric would have the same information as when someone reports the time varying E/I balance due to a sensory input? Think about what the rates of minis represent, think about what the amplitudes represent. And think about whether if you did some math on these, whether they would answer the question you hope to answer.
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Hello everyone! I'm considering long-term/ extended puff-application of compounds onto neurons in brain slice electrophysiology. This would be better for me than bath application because I have limited compound amount, and would I need to hold my cells in whole-cell, thus I want to keep them as short of a time as possible.
I've been searching the web for papers where they use the puff application in this way, but I cannot seem to find any. I know that it should be possible based on the fact that the picospritzer can potentially take 99.9 minutes to release all of the liquid in its pipette, but I want to know if it is feasible.
Any help/ examples would be greatly appreciated!
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Hi Emily,
I did some experiments where I delivered a drug by puff-application for a few minutes:
See the ‘Local application of nAChR agonists’ section in Methods. Could ‘Local application protocol II’ be what you’re looking for (see fig 3a for example)?
There, I used pipettes with a tip of about 2um diameter (which is probably close to the 1 MOhm resistance Jan Tønnesen suggested) connected with silicon tubing to a 1 mL syringe via a manometer. Filtering the puff solution before loading the pipette was enough for me to avoid those frustrating blockages Jan mentioned.
This method worked for me because I needed to limit movement of the tissue when puffing, and the syringe allowed a very ‘soft’ onset of the puff and not too high final pressure (few tens of millibars). However, if you are interested in the precise kinetics of the postsynaptic response, the picospritzer’s crisp, immediate onset and offset is certainly preferable.
All this said, I think those Baby-Bee syringes Enrique Soto mentioned would indeed also be very suitable.
Good luck!
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I am looking to study neurons of the bed nucleus of the stria terminalis. I have seen reports using coronal sections. Others present the distinct nuclei in transverse sections.
What is the best way to collect slices for the study of the BNST?
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I appreciate the question but this is not my area of expertise, I am a clinical neurologist
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I've recorded from neurons in the DMH of the hypothalamus. My evoked currents from my control and stress groups (unpaired t test) also did not pass the normality test, but were corrected with a 1/y transformation.
My paired pulse ratios are also not normalized. I don't expect either my evoked amplitude or paired pulse ratios to be normal but for statistical processes, I understand that a transformation is best.
Is this the right way to go? To transform 1/y for both my evoked and PPR? Because now in a graph, my PPR data is doing to be different than from other studies who present it as simply PPR, not 1/PPR transformation.
Additional information:
My protocol is:
5 min baseline (with 2 evoked currents 50 ms apart)
High frequency stimulation (HFS) Twice at 100Hz for 4 seconds, 20sec apart.
25 min post HFS (with 2 evoked currents 50 ms apart)
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Why don`t you try non-parametric Mann-Whitney test? In addition, it appears to me you are comparing more than two.groups, and in that case you cannot use t test nor MW. For that matter, non normal.data can be analyzed by kruskal-wallis and a post-hoc (i use nemenyi).
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I am concerned about vibration (what if it kicks in during a recording?), electrical noise, and actual sound (is it unbearable?). Thanks!
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Hi Argentina, thanks for this question. Most helpful! :-)
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I am trying to build an ephys rig with multiple amplifiers and use the WinWCP Whole Cell Electrophysiology Analysis Program to acquire the signals. In order to do so, I was planning to use a National Instruments PCIe-6353 analog to digital converter rendering 16 channels. I have previously tried the PCIe-6351 board with this software and it worked perfectly but I was wondering if there could be any compatibility issues with the PCIe-6353. Any suggestions will be greatly welcomed!!!
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Hi everyone, thanks for your feedback! As Javier Zorrilla de San Martin suggested, I have contacted John Dempster. He was extremelly kind and offered different solutions. Basically, he said I should be able to fully operate up to 4 amplifiers due to the limit of 4 analog outputs in these NI cards. In order to connect more amplifiers, one of the 4 outputs should be routed to the extra amplifier stimulus inputs. He also offered to upgrade WinWCP to enable the support of more than 4 amplifiers, which is the current default.
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I’m having some problems patch clamping iPS neurons as well as with embryonal mice neurons. They are anywhere from 20-40 days old and all have the same problem. Some of them have a smaller RMP (around -20mV), but even the ones with a more negative one ( around -40mV) have the same issue.
It is possible to seal them as well as to open them relatively easily, but then, when current-clamping, they show no action potentials and, while voltage-clamping, there is no sodium current?
Of course that without sodium current, we can’t expect an action potential, but what could be causing this absence of sodium current? We’ve tried neurons with different ages, to exclude them being too immature or too old already. I’ve also thought about the RMP not being negative enough, thus inactivating the sodium channels, but I’m guessing that even at -40mV there should still be a significant portion of them on the resting conformation.
What do you think the problem could be?
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Dear Nuno,
You cannot perform voltage clamp and current clamp at the same time because to register the Na current you have to eliminate all the other currents and you would not see action potentials properly. So you have to perform an experimental series with current clamp to study the action potential discharge and another series to study the isolated Na current.
The solutions we have used are shown in
Valdés-Baizabal C, Soto E, Vega R (2015) Dopaminergic Modulation of the Voltage-Gated Sodium Current in the Cochlear Afferent Neurons of the Rat. PLoS ONE 10(3): e0120808. doi:10.1371/journal.pone.0120808
You may probably test using neurobasal culture media.
Best Regards
Enrique
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Hi!
I've recorded from neurons in the hypothalamus with DNQX and Picrotoxin in my aCSF bath with the purpose of investigating signalling pathways (using HFS).
To get the most from one cell, I've also recorded Action Potentials in I clamp, using depolarizing steps. In this way, I've manipulated the membrane potential and triggered APs to fire.
Because these are not spontaneous or evoked inhibitory or excitatory currents, but are action potentials instead, can I pool cells that had been exposed to DNQX and Picrotoxin together for data analysis?
Any input is welcome.
Thank you,
Tenea
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If I understood well and in my opinion, for the purpose of statistical analysis and publication, you should perform separate analysis for those neurons treated with picrotoxin (as the GABA antagonist) and those treated with DNQX (as the AMPA antagonist). Although APs seem somehow identical and seemingly inconsequential in some experiments, the mechanism of action and their role in generation and propagation of APs and then fEPSPs or IPSPs are rather different in terms of channel activity and resultant synaptic activity. In particular when you use two antagonists which act differently and on different receptors.
You may analyze the recording and stimulation response of both separately and then compare them against each other though the later depends mainly on your setup, hypothesis/goal and recording/stimulation parameters. This can provide a better and conclusive insight regarding your common goal for different types of receptors and treatments too.
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Hi, I am planning to do some immunofluorescence with spare brain slices (400 uM thickness) from my electrophysiology experiment. Ideally I want to do the immunofluorescence 1 month after fixing the slices in 4% PFA o.n.
I wonder if I can keep the brain slices in 4% PFA at 4C or any other solution during one month without altering the morphology or the antigenic sites.
Many thanks in advance.
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Hi Chloe Hall, thanks a lot! Previously, for acute brain slices (400 um) I did overnight fixation with 4 % PFA but it is great to know that I can reduce it until 1h.
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What is the best commercially available electrophysiology system for chronic neuronal recording in awake animals and why? I listed some contenders below in alphabetical order. Of course there may be others that I am not aware of.
A-M Systems
Blackrock Ephys
Cambridge Electronic & Spike2
NeuralLynx
Plexon
Smart Ephys (Division of Harvard)
Tucker-Davis (TDT)
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Thanks for your note!
We will use this system to record extracellularly but have no immediate plans to record from single cells. I see some systems use amplification at the head stage while others do not.
I am open to either buying or writing software, but purchasing seems faster.
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I'm referring to the overlapping sigmoidal curves that are used to describe the gain-of-function mechanisms. I understand the activation curve, but I can't seem to get my head around the inactivation curve. Any help at all would be very much appreciated, whether that is an explanation or pointing to one in the literature. It would also be helpful if you could explain the holding potential, depolarising voltage steps, and pre-pulse vs test pulse. Thank you in advance!
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Did you get a satisfactory answer to this, or do you still need one?
The best explanation I ever found was by Peter Backx on youtube. He goes through both activation and inactivation on his channel. This is the link to his inactivation one. Let me know if you need anything else cleared up.
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Hi there,
Recently I have been facing an issue where I would get a nice gigaseal with resistance hovering around 1.5-16 gigaohm, but when I am recording channel activity, for 2-3 minutes the baseline would stay flat and stable with Channel openings, but after that the baseline would become unstable and start drooping. I have attached a image of what the droopy recording looks like. No matter how many cell I try it's the same thing.
After it starts drooping I would check the seal again and I can see that the gigaseal is still there intact.
Can someone kindly tell me why this happening?
Our solutions are sodium free and don't have any drugs in them. This was recorded at -50mv in cell attached voltage clamp mode.
Thank you in advance.
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Hello Sayeman,
if your seal is stable and maintains GOhmic resistance, then this drops might be channel openings themselves (probably potassium channels). You could check this by clamping the patch at 0 mV (which, if your K concentration is high should stabilize the seal) and at a positive potential like +80 mV which should make these drops go upwards.
Anyway, a big issue that I see in the picture you attach is that you have a masive hum (60 Hz) that you should eliminate for optimal recordings. Do you have any other sample?
Good luck!
Diego
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I am curious about two specific things:
- Why do pseudounipolar neurons have one axon (as opposed to a dendrite + axon like multipolar neurons)? How does this structure reflect sensory function?
- How do potentials propagate through the axon? Since there is no axon hillock for summation, does that mean no summation occurs? Is there still a threshold potential that needs to be met? Or does every graded potential get transmitted through the axon?
Can someone familiar with any of these questions help out or provide a resource I can refer to?
Thank you!
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Hi Sehej
Pseudounipolar pattern of sensory neurons acts as low-pass filtering, potentially regulating sensory information reaching the spinal cord. Thus, by impedance mismatch between membrane point in the vicinity of T-junction, this has been recognized as a site where spike propagation may fail.
As for action potential propagation through the axon, this is a more broad question. Actually, in a really simplistic summary, sensory neurons have "transductors" (specialized proteins that converts physical energy - thermal, mechanic, chemical - into electrical signal) in their peripheral end; these transductors creates a "generator potential", according to specifical thresholds. If, and only if, these generator potentials reach some specific area in the membrane with a larger density in voltage gated channels (as Na channels) within these second step threshold, an action potential (spike) is generated and conveyed until the "T-junction" filter described above.
Some references for better comprehension
1 -Al-Basha, Dhekra, and Steven A. Prescott. "Intermittent Failure of Spike Propagation in Primary Afferent Neurons during Tactile Stimulation." Journal of Neuroscience 39.50 (2019): 9927-9939.
2 - Sundt, Danielle, Nikita Gamper, and David B. Jaffe. "Spike propagation through the dorsal root ganglia in an unmyelinated sensory neuron: a modeling study." Journal of neurophysiology 114.6 (2015): 3140-3153.
Regards,
Tiago Avelar
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I’ve been unhappy with my slice quality lately and I’m looking to improve slice health. I’ve tried various times/variations of simple extractions and also transcardial perfusions. I just can’t seem to find the right combination. I’ve tried one to three minutes of perfusion with carbogen bubbled acsf without fantastic results (I clamp the abdominal aorta). Also, I was wondering if anyone had success using HEPES in a holding solution rather than just holding the slices in recording ACSF. (I’m doing recordings on 90-120 day old rats, by the way.) Any input would be really appreciated!
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For older mice (12+ weeks), we've had good results using an NMDG-based slicing and recovery procedure.
Unlike in the JoVE video, we slice with a vibratome and so the slicing takes longer, but otherwise our procedure is the same.
At the Allen Institute, they use this for human tissue, and so there's no reason to think that it shouldn't work with rats too.
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Electrophysiological testing are non-invasive and helpful tools to obtain objective parameters of the funcional status of the visual system. ERG and PERG are applicable to evaluate retina and can be useful to evaluted melanomas whereas VEPs are very important to monitor the optic nerve in orbital tumors. However, such exams are poorly explored in eye, orbit related neuro-oncologic settings.
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i agee
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I know for fast EPSC non-NMDA channels are involved and similarly, slow EPSC is mediated by NMDA channels. But what's the actual mechanism underlying these processes?
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The difference in kinetics of AMPA and NMDA channels is due to the difference in ... kinetics of the underlying ionic channels.
AMPA channels are chemical -gated ( by glutamate) channels that open very fast (<0.5 ms) and close very rapidly (<2-3 ms). NMDA channels are chemical -gated ( by glutamate) channels that open much slower (~5 ms) and close even more slowly (20-100 ms).
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Does anything happen to the reversal potential of AMPAR-activated ion channels when you use a CsCl internal solution? (i.e. ~140mM CsCl) I am aware that excess internal chloride is used to create an outward negative current when GABAa channels are activated, and that Caesium is used to block potassium channels. Do either of these facts change the E rev of AMPA?
Thanks
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A CsCl internal solution is often used to record EPSC through AMPA receptors. AMPA receptor channels are about equally permeable to Cs as to K, so that the reversal potential remains at about 0 mV. Cl does not really interfere, but I suggest to block the other receptors to better isolate the AMPA EPSCs. A GABAA receptor blocker is highly required to eliminate spontaneous and miniature GABAA IPSCs, because with high intracellular chloride they revert near 0 mV as the current that you want to measure.
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I need to induce LTP in hippocampal acute brain slices (rodent) and measure extracellular fPSPs.
Different bipolar stimulation electrodes available on the market have different impedances (0.1 MOhm, 0.5 MOhm, 1Mohm, 1.5 MOhm, and 2 MOhm) and I am not sure about the most suitable (no info from the customer service…).
Could anyone recommend a particular impedance based on experience?
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Hi there. I use 0.1 MOhm tungsten electrodes. Using 0.5 MOhm or higher impedances didn't give good ltp results with my recordings. Good luck.
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We Have a Zeiss Axiovert S100TV scope. It's a inverted microscope. I was wondering whether I can set this up for recording fEPSP from hippocampus?
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Ha ha ha. Actually, in all my previous labs the rig was ready to use. I learnt it and started experiment. Now, here I am doing it from scratch. So when I saw the slice. It looked great and I used the bipolar electrode which is really dark. So, with 300 micron slice I was able to see the area. So, I thought to set this up then see. Placing the electrode in the hippocampus is very easy. But, the glass pipet would be little bit tough. Will keep you posted what solution I make on this. ha ha.
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We are going to start a study without intervention, just electrophysiological exams.
Do We have to register it at clinical Trials?
thanks
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Hello Roberto,
Regarding your question, registration of observational studies not mandatory, but I advise you to register it. You can follow the instructions in the attached link: https://bit.ly/2t130EQ
Best wishes.
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Dear All,
I am wondering, could anyone help me out with a manual or datasheet with specs for this good ol` beast the RK 300 from Bio-logic? It would be very helpful and very much appreciated!
Thanks in advance for taking the time!
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We did have photocopy manual for biologic 400.
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Hello!
We are planning to publish a script for automatic firing-pattern analysis of .abf files in MatLab.
To make this script as useful as possible we would like to know your opinion.
Our short survey is aiming to estimate an interest in such a script, and, to improve its functionality by collecting the list of most user-analyzed features.
We will appreciate a lot if you could find 5 minutes to finish this survey!
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I didn't complete the survey as it seems like it would only be relevant for people doing intracellular recordings, while I focus on extracellular recordings, so I was not sure if my responses would be relevant for you. This issue should be clarified at the beginning of the survey.
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The most accepted definition for RRP is " a small subset of the many vesicles in presynaptic bouton that is more readily released than other vesicles which are in the recycling and reserve pool", and RRP consists of "docked" vesicles. Well, can we increase the number of docked vesicles in the presynaptic terminal? If we do it, what is the physiological mechanism underlying this increment? Does this increment depend on the increase of intracellular Ca2+? Does the increment of the size of active bouton lead to the increment of the size of RRP?
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