I am having trouble to replicate one of the main postulates about the expression of late LTP, concerning its dependence on de novo protein synthesis. I performe fEPSP recordings on the Schaffer collateral to CA1 synapse in rat hippocampal slices. Stimulate with 4xHFS (100 Hz, 1 sec), every five minutes. However, I cannot see an effect using anisomycin 30 µM on late LTP, even if I preincubate the slices in anisomycin for 2 hours before the experiment.
Villers et al. (PLoS One. 2012;7) already reported no effect of anisomycin or cycloheximide on late LTP. In this paper they argue that the protein-synthesis hypothesis for development of late LTP should be reconsidered.
What do you think? Do you have any idea about why these experiments are not working?
Thank you in advance for your help!
Diego Fernández
Building on to what Gabriele mentioned:
How often are your test pulses? That matters too, see Fonseca studies listed below.
At slow rates of test stimulus, I had to wait many hours. Others get faster effects with more frequent test pulses.
Redondo RL, Okuno H, Spooner PA, Frenguelli BG, Bito H, Morris RGM (2010) Synaptic Tagging and Capture: Differential Role of Distinct Calcium/Calmodulin Kinases in Protein Synthesis-Dependent Long-Term Potentiation. J Neurosci 30:4981-4989.
Also, I advise that you keep a non-stimulated pathway as control at all times so you can monitor overal slice excitability/health.
Fonseca R, Nagerl UV, Bonhoeffer T (2006) Neuronal activity determines the protein synthesis dependence of long-term potentiation. Nature Neuroscience 9:478-480.
Fonseca R, Nagerl UV, Morris RGM, Bonhoeffer T (2004) Competing for memory: hippocampal LTP under regimes of reduced protein synthesis. Neuron 44:1011-1020.
Fonseca R, Vabulas RM, Hartl FU, Bonhoeffer T, Nagerl UV (2006) A Balance of Protein Synthesis and Proteasome-Dependent Degradation Determines the Maintenance of LTP. Neuron 52:239-245.
Good luck!
How do you prepare your anisomycin? You may be oxidising it.
If that fails tets emetine and puromycin to confirm the mechanism is protein synthesis dependent.
I prepare anisomycin in DMSO, normally 60 mM aliquates, to be dissolved in ACSF to 30 µM. I tried other protein synthesis inhibitors as cycloheximide, and although is seems to be more effective, the results are still not very reproducible.
Thank you very much for your helping contributions!
Best regards,
Diego Fernández
Actually, you didn't induce a strong L-LTP. From, your figure the potentiation is less than 150%. In my hand, only one train 100Hz 1s induced LTP can last for three hours like your figure.
You can optimize your LTP by increasing the temperature to 28-30. And don't stimulate the slice very often, 0.033Hz is OK for baseline.
Something I noticed in my experiments, if your aliquots are older than two weeks, anisomycin doesn't work very efficiently. And, as Mao Yeh pointed out, your L-LTP doesn't look very robust.
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