Hi guys,
I'm a little bit in trouble with LTP on CA1. I performed some experiments inducing LTP after HFS (1 for 100 stimuli at 250Hz). This protocol is able to induce a strong potentiation of the synapse. My problem is that the potentiation after the induction is not stable and the response continues to increase without reaching a plateau. What do you think the problem is? I recorded extracellularly fEPSPs from hippocampal brain slices (400uM thickness; flow rate of about 2 mL/minute).
Thanks in advance for your advices.
Luca
Hi Michel, Thanks so much for your answer. I do not cut the CA3 and i do not block GABA. I record at room temperature in acsf with 1 mM of Mg and 2 mM of Ca. I used 250hz protocol because i used this before and seems to work very nice. The grey trace is the same experiment in KO mice and the black One is the WT. Before starting the recording i wait at least 30 minutes when in my opinion the response is stable. Before recordings i performed i/o curve and after i use the 50% of maximum response to do the experiment....maybe i have to wait more than 30 minutes before recording.
Thanks so much.
Luca
Hi All
I found that the recovery process in the holding chamber for me was one of the most important step I use to wait at least an hour then transfer to the recording ochamber let the slice to equilibrate for at 15 minutes without stimulation and recording (recordings at 28-30°C). After 20 minute baseline (stimulus every 30s) Apply HFS (Done teta burst or even 2 trains at 100Hz for one sec each 30s apart). Why did you chose this LTP induction protocol? Is there an experimental reason behind this? Your PTP looks small after your HFS Could you attach example of traces recorded during baseline at the PTP and at 70 minutes after HFS. Could be that you have lost the GABAergic inhibition and that your slices has become epileptic. How old are your mice?
Good luck.
@Michel: I record in submerged recording chamber and I apply stimulus every 20 seconds. I'm pretty sure that my electrodes are immobile during the recordings. I was thinking to try another stimulation protocol, maybe 250Hz is too strong :)
@Charles: maybe we have a misunderstand, when i say that i wait at least 30 minutes i mean that when i put my slice in the recording chamber i wait 30 minute before to start the recording but after the slicing procedure i wait 2 hours more or less. I used this protocol because i used this before. What do you mean with PTP? mice are more or less 3 months old.
Thanks so much
Ok, the first fEPSP after HFS some times increase so much and sometimes decrease , but after 2 or 3 responses the amplitude and the slope increase in all the experiments, but the response don't stabilize, i can try also to block GABA before recordings but in CA1 usually the GABAergic transmission is not a problem to induce LTP.
Thanks so much again
Luca
Under the microscope how does your slices look, bright and shiny with nice cell line?
In the CA1, if the GABAergic tone is damaged, you do get epileptic EPSPs that look spikey (increased amplitude or slope and could explain what you observed) and the EPSP duration is also increased. In normal condition, following HFS your EPSP should increase. Do you have any traces recorded in these slices? One stupid question have you checked your temperature probe (battery)? Don`t put picro or bicculine it will make your EPSP epileptic, not in the CA1. It works in the dentate but not in the CA1
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