It would help a lot if you could tell me the anesthetic used and the synapse your studying. In my experience, LTP failures are most commonly due to the stimulation intensity being too low, esp. with pentobarbital anesthesia. Are you getting "population spikes"?

Dear Dr Brain,

Thank you very much for the response.

We are using urethane anesthesia.

We are recording from the CA1 region, stimulating the Schaffer collateral pathway. Yes we are getting population spikes. We tried up to 3 10X trains to induce LTP, but no success

Please let me know if you require any additional info

Thank you

Pradeep

That situation should give you potentiation with little trouble. Urethane doesn't enhance GABAergic inhibition too much (like pentobarb does), and getting a spike suggests your stimulation intensity is high enough.

Perhaps you could try "theta burst" stimulation?: five 100 msec trains of 100 Hz, delivered every 200 msec for 1 sec. This induces robust LTP in our lab.

We also successfully induce LTP using “five 100 msec trains of 100 Hz, delivered every 200 msec for 1 sec” as Brian E Derrick wrote.

Are you using a bipolar stimulation electrode? 

Suggestions:

1 - avoid the stratum moleculare

2 - Too thin, bipolar concentric electrode, could make harder to recruit enough fibres to induce LTP.

Good luck

Thank you Dr Zuner A Bortolotto and Dr Brian E Derrick

Can anyone advice me how to measure EPSP amplitude using spike 2 sofware

I am also facing similar problem, we use isofluorane or halothane gaseous anesthesia

We are working on male Wistar rats of 4 months old weighing ~250-300g

I am obtaining 400um transverse sections by isolating the hippocampus

I am trying to record from CA1 synapse and stimulating the Schaffer Collaterals using bipolar cluster electrode

I am getting very good EPSPs with nice fiber volley and pop spikes at higher currents, my I/O curves are also good but I am not able to induce higher potentiation (greater than 50%) in my slices by neither TBS nor HFS. Most of the times, I am getting around 25-40% potentiation and very rarely 45-50%. I tried all possibilities of HFS like single train vs 4 trains or TBS with 10 bursts, 20 bursts at 100Hz with 5Hz  frequency or repeated T0 4 times with 10 bursts separated by 10s interval.

I tried using both sucrose based cutting solution and normal aCSF solution for obtaining slices, both at 4 degrees temperature. I am recording at room temperature 

aCSF Osmolairty is around 315 mM, CaCl2 2.2 mM and MgCl2 1.3 mM and I don't add any antagonist or agonists to the circulating solution

Can anyone help me in troubleshooting 

Hi Raghava,

Few things.

Make sure that the distance of the stim and recording electrodes  is not too far. Try 0.5 to 0.4 mm  far a part from each other and see if it improves.

Also, if you are recording in submerged slice, then it is expected less potentiation compared with interface configuration. 

Important:  keep the slice at least at 29 G. centigrade  (plus / minos 1). 

Good luck

Zuner

Dear Zuner A Bortolotto

Thanks for the reply

Yes we are using submerged chamber setup for recording without temperature control. Today I changed the stimulated electrode to new one and to my surprise the shape of the EPSP is more narrow  and amount of potentiation dramatically increased to 45-50% with TBS. I need to check these results by repeating the experiment couple of times.

We refereed your paper in Current Protocols in Neuroscience 

Once again thanks for the help and I will update my recordings outcome after couple of days.

Hi Raghava,

45 - 50% potentation in submerged slice is fine. 

Important to keep the stim electrode clean. We wash our stim electrodes every time we use by removing it from the slice but leaving it  within the recording chamber (that is in the water) for the duration we wash the chamber with purified water.

Do you use concentric or parallel (twisted) bipolar stim electrode? 

Well done it looks like you are getting things to work well.

Zuner

Dear Zuner

I am using bipolar cluster electrodes with 25um tip made of Pt/Ir, we have concentric bipolar as well but I felt using Cluster electrodes easy as they damage the tissue less and you can adjust it to fine tune the response.

I too leave it for washing after experiment with distilled water. People suggested me to run the tubes with sodium citrate to get rid off salt precipitate. Does it really help ? If, is there any defined composition of the sodium citrate solution ?

I will repeat the today's experiment couple of times and get back to you. I need to get the consistency before I start the actual experiment.

Thanks for your kind support

Dear Raghava,

I don't have experience about cleaning the electrodes with "sodium citrate". But I guess it could help once the SC is also washed out. 

Wishing all the best.

Zuner

Dear Zuner

I observed that the maximum output value of a slice varies even within the same animal. Some slices show maximaum slope value around -0.350 mV/ms, some around -0.750 mV/ms and some even goes beyond -1 mV/ms. hence my stable baseline values varying from slice to slice and animal to animal. is this variation normal ? 

Dear Raghava,

Yes, it is normal. There are many factors that would/could interfere/ influence the quality and magnitude of the sweep output. For the sound of what you are saying, you are doing well. So, now you need to concentrate in your experiments and with time you can become an expert. Good luck.

Zuner

Dear Zuner

This is the amount of LTP I got from 2 animals, 4 slices. Initially I was using sucrose based aCSF and the amount was 30%. This data is from normal aCSF cutting solution. Hope I will get the consistency and get good data from my electrophysiology experiments

Many thanks for your help

Dear Zuner

Sometimes I am getting this huge pop-spikes in my recordings (see the attachment), is there a way to avoid these. I am trying to place the recording electrode as far as I can from the Stratum pyrimadale but these are coming frequently. The main problem is, it is distorting the "decline phase" of the EPSP and obviously its shape. I feel, this may give me erroneous results if I compare the same with my experimental animals (Epileptic) for coastline analysis etc. 

Any comments on this ?

Dear Raghava,

The pop/spikes could be a result of few things such as: Quality of your slices (how health they are), Oxygenation, Over stimulation (stim intensity too high), position of your electrodes etc...

Maybe is worthy you trying the following (see the attached fig below).

Avoid this far end of the CA1. That part of the CA1 contains the temporoammonic input into CA1 and plasticity works differently.

Cheers

Zuner

Dear Zuner

The stimulation intensity was within the control (less than 80 uA). Electrodes position was varying as I was trying to get good EPSP shape. I am not sure of slice health quality but it took sometime for me to take sections last time. Right now, I think I placed the recording electrode in the position you are not suggesting. I will get back to you if anything is required.

Thanks for the response

Dear  Raghava,

Make sure you leave the slices to rest for at least 1 hour after you cut the brain. I also leave the slice to rest in the chamber before I try to get EPSPs. 

Cheers,

Zuner

I keep ~2h incubation in the slice keeper before start obtaining recordings 

Dear Zuner

My experiments are going well but I am having a strange data from experimental animals (Epileptic). They are showing highly impaired PPF ratio, no PTP after TBS and subsequently no LTP, occasionally I am seeing 10-20% potentiation (n=3/16). Most of the slices did not show any potentiation. The slope value is coming back to baseline immediately after the TBS (the first value itself !). I assume that this may be due to chronic epileptic condition (60 days) but is it really possible ? So far I have done 5 animals and roughly 16 slices. There is no POP spike contamination after your inputs.

Any comments on this ?

 Dear Raghava,

Just like that it is hard to follow your descriptions or understand what do you mean.

Would be possible for you to send some figures (plots)  with traces sample (that is the EPSPs responses you are recording)?

So I can try to understand what do you mean and eventually say something that could be of help to you?

Cheers

Dear Zuner

Please see the attached image. Whole last week I had done around 6 epileptic animals slice recording with slice number n=16. Most of the slices were showing similar response like in the attached representative image. The I/O curves look normal. After plotting I/O curve, selecting baseline current stimulation, I am doing paired pulse facilitation study by delivering 2 pulses at various intervals ranging from 40ms to 240ms. After PPF, I am allowing the slices for baseline recording followed by TBS after 20 min. As highlighted in the image, there is not post tetanic potentiation (PTP) and subsequently long term potentiation (LTP). I am not sure why the slices are undergoing The lack of PTP and LTP is puzzling me. Is it due to PPF study in the same slice or severely impaired hippocampal synaptic plasticity in epileptic animals or any other phenomenon ? I observed similar results sometime in normal control rat slices but the no of such failures is less compared to Epileptic animals. Do you think its an experimental error ? (Like placing electrodes too close etc).

However, 4 slices out of 16 showed PTP with reduced LTP compared to controls. My question here is, how do you select data for interpretation ? Do you select data that has PTP & reduced LTP or no PTP & LTP or both ?. I am feeling that if you selectively pick slice data that has PTP & reduced LTP, it leads to investigator bias. Is there a unbiased way ?

Generally what is the success percentage of inducing LTP in field recordings ? I read from papers that HFS is unreliable compared to TBS in inducing LTP so, I switched to TBS recently and found to be more reliable.

PS: The recordings in attached image is digitally low pass filtered at 200 Hz, hence its smooth without fiber volley.

Dear Raghava

Thank you for sending a picture of your screen.

Few things to try:

1-      I think you should try to record at low pass filtered at 10 or 15 or 50 Hz, to see what happens. Your trace does not looks normal to me. Look too much filtering.

2-      Your PPF responses look too small. I mean the facilitation is too small compared to ours. So it is possible that your problem sits in the stim vs recordings.  I am not sure what could be. So start by changing the filtering as suggested above.

Would be possible to send me a photo of your electrodes positioning. A assume you use a microscope to help you to position the electrodes. So you could use a Mobile (cell) phone and take a photo using you microscope. It is a bit fiddling but it is possible. It will look like the attached photo.

It would help me to understand better your set up and maybe to suggest something.

PPF interval should be kept around 50ms. 40 or 50 or 60ms - see which one gives you the best increase.

Dear Dr.Zuner

Though I am getting good recordings, I am still concerned about the probability of getting an LTP on particular day (in Normal controls). I am not getting LTP every time I do the slicing, however I am getting good EPSPs and acceptable I/O curves. I am sure my slicing quality is improved as evident from the clearly visible pyramidal cell layer. Most occasions I am seeing big fiber volleys ( but less than EPSP amplitudes) and a drift in decline phase of the curve (more coastline). Any suggestions from your side to improve slice quality if its the issue ?

I am taking slices at 4 degrees using vibratome. Decapitation ~1min, isolation of hippocampus ~2 mins, slicing 8-12 mins. Total time = ~12-15 mins to get all slices. 

I have one more query, is it fine to perform PPF studies in the same slice or I should consider a new slice exclusive for these studies ? I observed POP spike in 2nd EPSPs in some cases. After the PPF study, without changing the stimulation, there is an increase in output as well (~10% increase).

Any comments ?

Dear Jagadeesh

Where are you employed the electrodes? What region of hippompus are you choose? The DG-PP or CA3-CA1 area of Hippocapus?

CA3-CA1 pathway

In this case, the positioning of the electrodes is very important. For us works when the electrodes are positined as follow: (concentric bipolar tungsten electrode) positioned in the granule cell layer of the dentate gyrus according to the co-ordinates from bregma (anterior–posterior [AP], 23.6; mediolateral (ML), 22.0). ARecording concentric bipolar tungsten electrode was positioned in area CA3 of the hippocampus (AP, 23.0; ML, 22.3).

For more details plea see this paper: An Interchangeable Role for Kainate and Metabotropic Glutamate Receptors in the Induction of Rat Hippocampal Mossy Fiber Long-Term Potentiation In Vivo. Wallis JL et al. HIPPOCAMPUS 25:1407–1417 (2015)

can you upload two sample traces, 1-min pre-tetanic and 1-min post tetanic of recording?

Dear Raghava Jagadeesh Salaka,

Could you please tell, did you manage to increase the LTP induction success rate in your experiments? And did you understand what was that thing with the absence of even PTP in epileptic experiments (which I also observe sometimes and have no idea, what's going on)?

Dear Denis Ashrapov

I am convinced that the LTP induction success rate entirely depends on the age of the animal. We discussed this part in our paper published in Journal of Neuroscience Research. See link below.

Regarding PTP, I did not find any difference in PTP between control groups and epileptic groups, the paper is due for publication and hence I could not share the data here. The image I posted above was when I was learning slice electrophysiology (2017). There were several issues with quality of slices and recording at that time. The traces were also not at all good in the above picture. So kindly ignore the one I posted above.

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