Hello everybody.
I have seen through publications that some labs have been routinaly able to induce very long-lasting in vitro LTD in young and adult animals. I would like to ask you if you can give me some details of the methods to achieve it.
I am trying to get LTD in my hippocampal slices from ~10 weeks old mice, but so far it has been almost impossible. I have been using both electrical stimulation (single 900 pulses at 1 Hz) or the application of mGluR2/3 agonist LY354740. I am stimulating the medial perforant path and recording fEPSPs in the dentate gyrus, at room temperature.
Does anybody have some advice to get stable LTD?
Thank you very much in advance for your contributions.
Diego Fernández
Can you get LTD in younger animals? And what are your cutting/incubation conditions---NMDG/sucrose, etc.?
In my hands, I've found that I need very healthy slices to get good LTD from older mice. Also, I've had better luck getting LTD using a paired-pulse LTD induction protocol vs. the 900 pulse/1 Hz protocol.
Hi Diego, It should work if do your the experiments in presence of Picrotoxin or bicuculline (the GABAR inhibitor must be applied during whole experiment). You will need to remove CA3 region to avoid epileptic activity. This trick also works for rats.
We haven't tried to get LTD in younger animals. All we have done was in this 10 weeks-old mice.
Although I found this paper (doi:10.1016/j.neulet.2004.04.056), when they described a clear age-dependency of LTD in mice, they say that this is mGluR-independent mechanism.
Here we really want to focus on the chemical LTD with the mGluR2/3 agonist LY354740. We tried to avoid the electrical protocol. So the question is more pointing to how to get robust and stable mGluRs group II-dependent LTD.
Do then your advice work for the induction of this LTD?
Thank you very much for your help.
Diego
Hi Diego, as you want to focus on chemical mGluR2/3 dependent LTD, together with all suggestions from our colleagues, I suggest you to have a look on the papers below. Both papers explore the roles of mGluR2/3 in the cortical – Temporo-ammonic path to CA1 region.
1. Ceolin L et al., Study of Novel Selective mGlu2 Agonist in the Temporo-Ammonic Input to CA1 Neurons Reveals Reduced mGlu2 Receptor Expression in a Wistar Substrain with an Anxiety-Like Phenotype. The Journal of Neuroscience, May 4, 2011 • 31(18):6721– 6731 • 6721
2. Hanna L et al., Differentiating the roles of mGlu2 and mGlu3 receptors using LY541850, an mGlu2 agonist/mGlu3 antagonist. Neuropharmacology 66 (2013) 114-121
Good luck.- Zuner
Hi Diego,
I would advice that you incubate your slice for at least an hour at 32°C with carbogen perfussion (95% of Oxygen & 5% of carbobdioxide). Also perform the experiment under the same condition. We used slice choper to prepare my slice less than 5-7 minutes from dicapitating the animal to cutting the 400µm acute slices in my previous lab.
After positioning your stimulating electrode in the perforant pathway and the recording electrodes in the dentate granule cells, you consider waiting for 20-30 minutes before taking the Input/Output curve, thereafter a stable baseline and then induction of your LDT protocol and you will definitely get a stable LTD up to 8 hours or depending on your choice.
You may consider taking a look at some papers from my previous lab, Sajikumar & Frey (PubMed). I hope you found this helpful and if you require any further further Information, don't hesitate to get back to me, thanks.
Good luck with your LTD induction protocol
Hi Diego,
I agreed with John Kodolo, slice preparation is very important. Double check your aCSF pH as well, sometime it affects your slice longevity.
I would suggest you to start with younger animal to optimise your protocol before you work with older animal.
All the best!
I am also doing LTD with 8-10 week old mice and i get the LTD very often but we keep the slice at 320C since after cutting. We use the same buffer for cutting and recording for further details check the publications from Frey,s Lab as John mentioned.
Hi Diego: I agree with the previous comments... but slice health (protection while cutting) and then temperature during storage and recording are very important. Recording at 'room temperature' is probably not ideal - I would suggest 32-33C.
Hello Diego,
We routinely achieve late-phase LTD in mice over 3 months that is similar in magnitude to animals of 18-20 months. These 2 citations may help.
Ahmed et al Neuroscienc 2011 Ahmed et al Neurobiol of Aging 2015,
You may try the paired-pulse low-frequency stimulation (PP-LFS): 900 pair pulses at 1 Hz (one pair each second), each pair is basically two 0.1 ms pulses separated by 20 ms interval.
Thank you everybody for all your comments.
It seems clear that slice health is a critical factor for the induction of LTD, so we will focus on improving our slice preparation and also we will modify our setup to be able to work at more physiological temperatures, following your advice.
These contributions and the papers you've suggested have been very helpful.
Thank you all so much again.
Best regards,
Diego Fernández
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