FIELD- POTENTIAL

[black]

  Hey guys, is there anyone doing field-potential in slices? I would like to know wether it is normal or not that many days I get absolutely nothing (in terms basicly that I don´t have a stable baseline lasting for all the experiment). I would like to rule out the possibility that it is due to some of my stimulators. I´ve read in some papers that some groups change polarity inmediately after one pulse so that it doesn´t get polarized. So far I´m not doing that ´cause not many people do it... I wonder if that could be the reason for my low "profitability". Thanks !!

[Thi]

Could you specify what kind of experiment you want to perform and what you mean by baseline? It appears to me you want to apply double-pulse stimulation to check for STP/STD?

[black]

   Hi, thanks for the interest Thi. I´m doing Long Term Potentiation in hippocampus slices (this is, I apply a high frequency tetanus and afterwards the response of the slice to the same stimulus I applied before the tetanus is bigger). For this I have to be sure for about 30 minutes before applying the tetanus that the response of the slice to a same stimulus is always the same (that would be the baseline). What I would like to know from other people who have done this experiments or just worked with field potential recordings is wether the recordings are always so "fragile" or it´s is just that I have to perfectionate something in my setup or experiment conditions.

p.d. No, no, it´s not double-pulse stimulation (I don´t know what that is ).

Thanks.

Black.

[Thi]

Ok so you're doing LTP (double-pulse-timulation would be applied for STP/STD). Considering your unstable baseline I would say that it could be bad positioning of both recording and stimulation pipette, especially if you want to record LTP at hippocampal mossy fiber synapses this is crucial because CA3 receives rather little input from the dentate gyrus as compared to CA1 from CA3. It also happened to me once that the optimal stimulation intensity is just not in the range of what you can adjust on the multiplier (i.e. because the steps are too gross if using old multipliers) though this was just a single case.
Apart from that anything could disturb your recordings, be it bad animal peparation (i.e. too much anaesthesia), an unsteadily fixed stage, too much noise from the electrical devices (check the grounding)... .
If you receive proper inputs once in a while it might be your handling (bad slices, pipette positioning etc.), if you never manage to get proper data it is most likely your setup, I believe. Maybe you should try neglecting the bad baseline and just go for the tetanus stimulation to see what the responses look like. Again, if you have at least bigger but unclean signals it should be your setup or pipettes, if you see no LTP at all well then it is your slice which lost consciousness for good;-).

I am sure this did not solve your problem but maybe it gave you some hints.
Keep me posted on things:-).

[Thi]

I forgot to mention the extracellular solution. Do you add Gabazine (or any GABA-receptor-blocker) to your aCSF?

[black]

   Hi Thi, ok, so you mean the paired pulse, witch is a form of stp, right? Well, lol, let´s go to the interesting part. No, I don´t add any GABA receptor blocker to the acsf. Do you use it to enhance triggering of LTP? It´s not that I don´t have LTP. After applying a tetanus for sure there is always a potentiation. The difficult part is that it doesn´t screw up (either it is because I couldn´t get an stable baseline or after the potentiation the slope of the EPSP goes lower than the baseline...) for the 4 hours I want to record.
   I´m doing LTP in Schaffer collaterals and.. I don´t know about the positioning. I understand a bad positioning of the electrode and stimulator could affect in terms that you find a good response or not but I do find it.
   About the optimum intensity I usually find one that gets me about an slope of 1 or 2 mV/mS. I managed (after a while...) that my stimulators apply the intensity I want. Can I ask you which stimulators ( I guess is the same as multiplier) are you using? Mine are kind of "home made".
   How much time did you have your slices resting? I leave them alone for 3 hours.
   I can register good signals (I don´t know.. I can get slopes of the EPSP of 8 mV/ms more or less) everyday so I kind of think I´m treating the slices fine.
   What I want to know the most is.. when you were doing LTP did you get valid recordings everyday or you had days in which you had to get rid of them?

   Thank you for your ideas !!!

[Thi]

Of course, I meant paired-pulse-ratio, sorry for the confusion dude . Reading that you want to record over 4h I assume you want to have both E-LTP and L-LTP. Well honestly I haven't done L-LTP so far and I guess your slice prep has to be different from what I did to allow for such long recording times. Moreover, I don't understand what you mean by the LTP "doesn't screw up...". If you mean it doesn't increase - well I have never heard of LTP getting stronger in vitro over recording time, it normally approaches the baseline asymptotically. If you mean the LTP does breaks down it is most likely your slice quality that gradually deteriorates or maybe even a part of your experiment (depedning on whether/how you manipulate), I believe.
To your last question, I have never done L-LTP, but one thing's for sure I've never managed to get valid recordings for anything on a daily basis .

[black]

   Yes, I meant it didn´t break down (probably I made up that phrasal verb )That´s the first thing I thought: my slices are not good enough, but I doubt it now. I don´t know, I´m starting to think the stimulators don´t work properly..
   It´s good to know that it is not very common to have valid recordings everyday !!
   Thank you for all your answer Thi.