Hi, at first sight your procedure seems to be ok. I usually wait two hours prior to recording. However, if i watch your signal it looks like too much "compressed" in the x axis. I use different software than yours, but the time scale i see on your x axis have too big steps in my opinion. if there is an option to change the scale factor in the time axis try to increase that and you should be able to have a better comprehension of your response. Moreover, if you look carefully you can see a kind of a slope after your stimulus artifact, but it will be easier to observe that if you enlarge the time window of your observation. To be more clear i'm attaching one of my recordings (please note the time window on x axis). if you can upload an image with a less compressed x axis maybe we can see something more!

Try to do that, i think you will see a kind of a slope in there after decreasing your scale factor!

There is something going seriously wrong with the electrical side of recording set up. I believe everything you are seeing on the screen is a stimulus artifact. Your stimulus artifact should be a very brief event, only taking up a few sample points. What are you using to generate your stimulus? What kind of stimulus isolator are you using? What are the properties of your stimulus (amplitude/duration). Until you're stimulus artifact is just a brief event, you're never going to be able to resolve an EPSP. 

  I think there is a problem in hardware.1.stimulus isolator 2. Amplifier or pre amplifier.

Thank you, Walter, William and Masoumeh.  For stimulation, I use bipolar tungsten electrode through ISO-flex stimulator.  Stimulated the inputs once every 15s.  Stimulus was given either at 10ms or 50ms for a duration of 0.10ms and voltage was 5mV.  Iso-flex is directly connected to the amplifier.  I use a pre-amplifier, Grass S88 for amplifying the signal and feed that signal into the amplifier.  Acquisition software is HEKA patchmaster.   I have also attached two images of my recent recordings - still having issues with the stimulus artifact.  Do you have any suggestions on how to reduce the artifact, so that I can start seeing a synaptic response?

For my experiences, you need to wait after cutting not less than 2 hours before to attempt any recording. I did not get reasonable output before. Usually after cutting  some "lesion potentials" arise due to the destruction of axons mainly this produce only a mess of strange waves or nothing. You need to wait some time for slice to recover cutting, repair what can still repair and  reach some new equilibrium.

Vito

Apparently you record a fiber volley ony.

There could be some basic reasons for the absence of synaptic potentias (with probabilities in descending order):

1. Missing Ca2+ from the ACSF.

2. Wrong angle in cutting slices.

3. Not yet recovered slices (wait a least 1.5h).

4. Dead slices (in that case you can still get a fv but not synaptic potentials). Try to prepare slices in cool ACSF.

Costas

Hello,

I agree with Costas: it seems like you're only recording the artifact and the fiber volley.

I'd suggest you to re-check your ACSF recipe (especially CA2+ and Mg2+ concentrations), to slice in ice-cold ACSF and to incubate the slices 1h at 30-32°C and the 30 min at room t° before electrodes placement.

Good luck!

Hi Costas and Andrei,  I have 4mM ca2+ and 4mM Mg2+ in ACSF.  Is this high molarity for field potentials?  I have not done slicing in ice-cold ACSF before, I will try it too.  After slicing, should all the incubations (elevated and room temperature) be carried out in ACSF too?  Thanks!

Hi Selvam,

4mM is too high for both Ca and Mg. In principle, however, it shouldn’t avoid the appearance of synaptic potentials. Try slicing in ice-cold ACSF and transfer slices to the recording chamber immediately. They don’t really need preincubation at room temperature. My experience was that preincubation makes not very healthy slices.

Hi Rajamani,

I would suggest trying 2.5 mM Ca and 1.25 mM Mg. 

I usually slice in ice-cold ACSF and transfer the ACSF-filled incubation chamber with the slices to a 32°C water bath for 1 h. Then, I turn off the water bath and let the slices to rest for 30 min more. Finally, after transferring the slice to the recording chamber I wait 5-10 min before electrodes placement.

Best!

Hello everybody,

Please help me. I see stimulation artifact and noise level is minimal for a field recording, but I get no field. I assembled this setup recently and I am trying to make it work. So far, I had no luck in finding fields. Please see the images attached, Can anyone tell me what the problem might be here please ?

Thank you so much.

Alice Bittar You have some serious issues. Even if your slices are 90% dead, you should be able to see the fiber volley, i.e. the action potential of the Schaffer collaterals (assuming you're in CA1). The fact that you cant see that means either your recording system is broken/not properly configured or you're not even in the slice. I know you can see the stimulus artifact, but you can't always use that to mean your recording side is fine, as you can get purely capacitive coupling.

Hi. I am having the same issue and was wondering what the solution was to your issue.

Thank you! Rajamani Selvam Alice Bittar