How long do you incubate the slices before the recordings? Try to increase the incubation time, the metabolism will stabilize. Some people incubate the slices for 3-4 hours for the same reason for the late LTP recordings. Also, I would record the baseline for much longer time than 10 min. We usually record 30-60 min of the baseline. In some slices it is simply impossible to get the stable responses. How good is your perfusion and oxygenation? What chamber your are using, submerged or the interface? I have noticed that the responses are much stable in interface chambers. Also, try to reduce the tissue polarization as much as you can - use platinum or platinum/iridium electrodes, and try biphasic stimulation instead of the monophasic one. The stimulator could be tested by measuring the stability of the voltage (with the digital voltmeter or the scope) on the resistor connected to the stimulator output instead of the electrode.

Hi,

Normally with 1:30-2 of incubation is enough, but is true that as much as you wait as more stable are the recordings. 15 min of baseline is enough but 30 min is optimal in cases when recording is not stable yet. If the recording does not stabilize in 30 min I would go for the next slice. Interfacambers have very good stability, but LTP can be done in both types of chambers. After doing the full protocol, at offline analyses, I would reject the experiments that continue to increase both at baseline and including the LTP phase, arguing a lack of stability. Also I would reject the ones that are not 'activated' by the protocol, meaning the ones that you did not get the LTP, always following some criteria, for example not having potentiation that lasts for more than 15 min. Cheers.

Hi,

For my experiments, 1-1,5 h incubation is enough. After placing the electrodes it is always good to wait for 10-15 mins, before starting to determine the input-output curve for each slice. It helps the slice recover from the possible disturbance of electrode placement. Afterwards you can determine your stimulation intensity and continue with your protocol.

You can check this effect by giving the same stimulation and checking the signal immediately after and 15 mins after the placement of the electrode. Cheers!

The incubation are important, you need "rest" the slice before to do ti the experiment. another impoartan details in the perfusión, the quality of the gas mixture are key, for stable signal and the flow must to be continue

good luck!

Decreasing baseline could be due to poor slice health, leakage from the recording electrode if you use a higher salt solution, or issues with bath temperature.

Increasing baseline---I would probably wait a bit longer than 10 min, but if is still "running up" then it may be some other factor....depending on if your incubation conditions differ dramatically from the recording conditions, it may be some time to stabilize between the two. For example, if incubation is at room temp and recording is at 32C, or if you are incubating in a modified aCSF, etc.

Ciao Giampaolo, Besides of all suggestions above, you may be able to get practical help from Prof RG Nistico, Dr Sonia Piccinin and or Dr Nicola Berretta, all working in Roma at Santa Lucia, Tor Vergata or at Sapienza. They have plenty of experience with extracellular recordings.

Unstable baselines have occurred in my experience due to many factors:

1-Faulty temperature control (fluctuations between 34-30 can alter field potentials dramatically), Check that the temperature is really stable.

2) Flow of the aCSF. High flow rates can cause mechanical problems. With a pump there can be subtle slice drifting with the ebb and flow of the motor. With gravity system,  be careful, rates can change dramatically if your source aCSF is in a small volume dispersed vertically  (like those popular  50/100 ml syringe caps). Generally high rates=big responses and vice versa. 

3) Re-equilibration of the slice for 1 hour is usually necessary if you mechanically moved the slice from a dissimilar holding chamber (especially if temperature and solution is different)

4) Drifting electrodes. Check the manipulator. Observe the slice, perhaps the electrodes are drifting due to inconsistencies in flow rate/slice subtly moving. If you are using a humidified interface chamber, droplets can appear on the electrodes that fall onto the slice causing mechanical instabilities as well.  

Good luck!

Thank you very much to everyone!! your suggestions were very useful and i have improved my recordings!