Ciao Alerssandra,

grazie del pensiero :)

Un abbraccio anche a te

Luca

Hi Luca,

Perhaps a few more details might be informative. You are perfusing the DHPG for 10 min, and after this do you wash it out? Do you include any inhibitors in your perfusing saline, like picrotoxin...etc?

One piece of information that was relayed to me about DHPG, although I have not seen references to support this myself, is that DHPG is a relatively labile compound, so the recommendation relayed to me was to always prepare fresh the amount required for the experiment of a given day directly from the lyophilized compound. Another thing to keep in mind is that there will be slices that just won't exhibit much if any DHPG-LTD; the overall LTD I've seen reported in the literature is rather subtle, ~20%.

Hope that helps,

Haider

Hi Haider,

my experimental protocol is 10 minutes of baseline recording, 10 minutes of DHPG 100uM and, after, wash out for 50 minutes to assess if LTD has been induced. Yesterday, that was my first attempt to induce LTD using DHPG i did not use any blocker but today i'm trying using bicuculline. If nothing changes today tomorrow i'll try using "fresh" compound.

Thanks so much Haider and have a nice day

Luca

update: I tried adding bicuculline (20uM) in the ACSF and at the moment seems that DHPG works (experiment in attachment)

 Hi Luca,

Our lab is regularly able to induce robust DHPG-LTD at SC-CA1 in mouse slices with both 50 and 75uM DHPG, without any antagonists on board.

I agree with Haider, it may be a problem with your DHPG stock/powder. You should see the sustained acute depression for at least the entire time DHPG is applied to the slice, not just for a couple of minutes as in your experiment

We've found that DHPG in stock solutions is very sensitive to freeze-thaw. We make up the stock solution, aliquot it, store at -20oC, and only use one aliquot per day, never refreezing the stock. These aliquots are normally only good for a couple of weeks max. Also, it take a while for DHPG to go into solution from powder, usually requiring sonication.

Making fresh DHPG stock solution from a new powder stock might help.

All the best,

James

Hi James,

Thanks so much for your answer. Effectively my DHPG aliquotes are one month old and also the powder (stored at -20°C) are 1 month old. I tried using aliquotes and also using the solution from the powder but without any difference, only in 1 slice i have been able to induce a stable LTD (not so huge, about 25% of reduction). Furthermore, i see from the Tocris website that DHPG is present in 2 or 3 different molecules. I used RS-DHPG, which kind of DHPG do you use in your lab? Another curiosity is about mouse age, how old are the animal that you usually uses for this kind of experiments?

Thanks so much and have a nice day

Luca

Luca,

To answer your questions: (1) We use S-DHPG and (2) we use male, C57BL/6J mice between 6-10 weeks old and still see around 60% depression with 50uM DHPG. I agree with Haider though, with a low N it's hard to make any conclusions as to whether or not you get LTD. How many slices have you tried now with not LTD?

Slicing/recording conditions could make a big difference. We use coronal sections, recording from the dorsal hippocampus using a submerged chamber. The ACSF is perfused at 2mL/min at 30-31 degrees Celsius. What are the conditions that you are using? Are you sure you are stimulating the Schaffer collaterals and recording in the stratum radiatum of CA1? Whenever we do not see DHPG-LTD it is normally because the stimulating and recording electrodes are improperly positioned.

Best,

James

 Luca,

How long are you allowing your slices to recover before recording?  I have heard that slices need to recover at least 3 hours before recording to maintain a robust response for DHPG-LTD.

Angela

Hi Angela,

for fEPSPs recordings i usually finish the slice procedure at 10.00 a.m. and i start the experiment after lunch, around 01.00 p.m. because in my experience, after 3 or 4 hours of recover, to find good responses are easier :)

Thanks for your answer

Luca

Hi James,

my experimental conditions are more or less the same that you use for slicing or recording procedure. I'm sure to stimulate schaffer collaterals and also to record from the stratum radiatum. The only difference is that i use RS-DHPG and you S-DHPG, maybe i'll try the second one.

Thanks again

Luca

Dear Luca, I had the very same problem in the past, using RS-DHPG I didn't manage to get a reliable LTD. Using S-DHPG solved the issue.

Wish you good luck!

Sebastiano

Hi Sebastiano,

Thanks so much for reporting your experience. I will try S-DHPG.

Have a nice day

Luca

A bit late, but I think I'll add my 2 cents: RS DHPG is a racemic mixture, while S DHPG is a pure S-isomer that works. It essentially means that you never know the amount of your active component in RS DHPG (you can estimate it's 50%, but it's only estimation).