Congrats, you got a nice record, and you have done good experiments, just go ahead.

it is normal to have some slices without LTP. You can also report the percentage of slices that show LTP.

Dear Diego

Your records are OK . In biological systems it is not strange to observe such kind of differences, however, you have to consider some points. It is important to be sure that in all of the slices you are recording monosynaptically; it seems that there are some extra-spikes in some records (e.g. slice 3). I recommend you to record from source instead of sink. In control situation, the percentage of slices showing LTP is usually more than 50%. I'm sure you can observe LTP in more percentage of slices in your next experiments.

Hi Diego,

Great!! its normal in electrophysiology and the recordings are fine and for early and late LTP we used different protocols.

for late LTP experiments (recordings for 6-8 hr) we used 3xtet 100 Hz 100 pulses and intertrain interval was 10 min and for early LTP we used 21 pulses 100 Hz instead of 100 pulses. The early LTP will completely decays to the baseline with 2-3hrs only so don´t worry if you want record it for long term experiments.

But some people use 1 TET 100 Hz 100 Pulses as early LTP but this will also give you a late LTP, which is protein synthesis dependent and long lasting atleast up to 6 Hrs (see sajikumar et al 2008, Learn.mem 2008)

so all the best

Thank you very much for your helpful comments.

Farshad, it is good to know that some discrepancy in the results using 100 Hz 1 sec is normal. I am glad to hear that the experiments don't look that bad! :)

Javad, I am measuring the slope so I think that I am avoiding these extra-spikes that are very evident in some slices, as you mentioned.

Thank you Binu for your advice. I have made new slices with 20 stimulations (n=8) and now the difference after 1 hour is significant (see pdf attached). However, do you think that this percentage of LTP (16%) after 1 hour is enough or is too small? Should I try experiments with 21 stimulations anyway?

Thank you so much for the time you have dedicated to this post.

Hi Diego,

I don´t know about what kind of experiments you want to do, we used the 21 pulse protocol, which was protein synthesis independent and we used this protocol for reinforcement studies and synaptic tagging (Frey & Morris) in vitro. If you could give me a just hind about this i can just you something.

Best

Yes Binu, I just want to validate a protein synthesis-independent, early LTP. I want a reliable protocol for further pharmacological evaluation. I need to be sure that this is actually early LTP. And actually, when I try 20 stimulations I don't have slices showing late LTP, what is fine, but the potentiation after one hour is not very big (16%), so barely significant compared to baseline levels. The problem is that when I try more than 30 stimulations I start having slices with long lasting (more than 3 hours) LTP, so I can't use them and be sure that is early LTP

In general, I want to try compounds and test whether they affect early LTP levels or they transform early into late LTP. I already have validated LTP with 4 trains 100 Hz 1 sec.

So the question is, do you think that this early LTP that I get with 20 pulses is good enough (in terms of amount of potentiation after one hour) to be used to study early LTP? or should I rather look for a protocol to achieve greater levels of potentiation after 1 hour?

Thanks a lot!

Hi Diego,

Great!! i think this is absolutely fine and we were using since 15 years the early LTP protocol and it is protein synthesis independent but only the thing was we were doing long term recordings 6-8hrs and for that its fine if it decays to baseline with in 2-3hrs. but if you use the same protocol as you tried better to record bit more than one hr. But if you do some reinforcement studies then you will compare between the groups and the 16% will become statistically significant .

Hope this will help you bit more and if you need something more don´t hesitate to write

Best,

Binu

Thank you so much for such a good help!

Hi Diego,

i forgot to tell you one more thing , we have performed all these experiments in rats but not in mouse, so please let me know if the 21 pulse protocol works.

Best

Dear Diego,

keep going gathering more data, with at least 20 good experiments (stable baseline before HFS and good fEPSP) you can make more robust conclusions about your results, and also is good to report the number of slices able to have LTP along with a cumulative probability of your induced LTP (showing the average and SEM is not enough).

Another important thing are your fEPSP, you must be more consistent with the position of your recording, you have a mixture of fEPSP recorded in striatum pyramidale and striatum radiatum, always try to get striatum radiatum because you can get the presynaptic volley, which can be used as a control for the stability of your recording.

Also is good to use different HFS and also TBS to explore all possible variations in pathways activation, take a look of the following paper: Raymond CR. LTP forms 1, 2 and 3: different mechanisms for the "long" in long-term potentiation. Trends Neurosci. 2007 Apr;30(4):167-75.

Buena Suerte.

You mention that you perform the experiments at "room" temperature. Is it controlled to within half a degree, or variable with the weather? Cooling the brain has a number of effects that alter neuronal activity, so a variation in room temperature from experiment to experiment could be one source of variation in your LTP durations.

My apologies if you have thought of this, and merely didn't mention the controlled condition.

Thank you for your answers!

Binu, I haven't tried so far 21 stimulations. We have made experiments with 20 stimulations (100 Hz) but the LTP is not very robust, and is back to baseline just before one hour after stimulation. Now I will try 25 stimulations, to see if I get an stronger LTP. Do you think it makes more sense trying 21 stimulations? For me it is hard to believe that just one pulse will make such a difference. What do you think?

Hola Carlos, I am trying hard everytime to place the electrodes in the right place, I look for the stratum pyramidale, which appears like a thin dark line... and stratum lacunosum.. and I place the recording electrode just in between them, in the half portion of stratum radiatum. I think this is right. The stimulation electrode is located in around 500 µm away, in the same position in str. radiatum. Thank you for the paper suggestion, it has been really helpful. I haven't read before about this LTP1, 2, 3 classification.

Hello Lynn, normally we work in a room with air conditioning. So normally the temperature is well controlled around 22 °C. Thank you very much for your contribution.

Hi Diego, would it be more appropriate to use higher temperature in your chamber, perhaps a better approximation of pseudo-physiological state? I would expect maybe 32*C would be ideal but maybe someone else with more expertise in LTP can weigh in on that parameter.

Dear Diego Fernández-Fernández,

Could you please tell, did you eventually manage to induce stronger LTP (>16%)? And was you able to increase the success rate of LTP induction (like >50%)?