I'm trying to come up with an LTP protocol induction(TBS) in CA1 pyramidal neurons which reliably lead to firing of neuron.
I'm using a theta glass for bipolar stimulation and the diameter of the tip is approximately ~40-50um. I'm interested to stimulate proximal synapses of SC.
when I set the baseline EPSP to be around one third of the difference between firing threshold and resting membrane potential, in most cases I won't get a spike although I can see the potentiation effect.
I tried different position for stimulating electrode when it's distant from the neuron (more than 600um) I can't usually get big EPSPs.
What is the best strategy to have spikes during induction?
Should I try blocking interneurons?
Dear Forouzan,
first a few questions
For theta burst LTP to work people usually use 2/3 of maximum (spike threshold) as stimulation. The spiking during induction is needed for the post-synaptic depolarization which allows the Calcium influx. I have even gotten LTP with only 2 burst of 4 stimuli each. See page 167 of my thesis http://m.tipharma.com/fileadmin/user_upload/Theses/PDF/Nils_Zuiderveen_Borgesius_T5-207.pdf)
best
Nils
Nils Borgesius , thanks for your response but I'm doing patch-clamp. So as I mentioned, I'm recording EPSPs. and I'm looking for a protocol which will guaranteed the neuron will spike during induction. and I want to stimulate SC synapses at 100um away from soma.
Forouzan Farahani, sorry I did not read carefully enough. If the cell needs to spike it has to be in current-clamp. If you have in voltage-clamp it cannot spike. But since you're recording EPSPs I suppose you do this. As I mentioned, 2/3 of the spiking threshold should probably work. However, in your case it is an (easy) empirical question. As you record the membrane potential during induction you can see if it spikes or not. You can try it in a few cells, adjust your protocol accordingly and even discard any experiment where the cell did not spike.
good luck
Nils
As mentioned by Neil, you can manipulate the stimulation strength to increase the size of your EPSP and hence spike once bursts at 100Hz. I would keep the tip of the stimulation electrode quite close to the dendrite to increase the probability of spiking/EPSP.
Canyou slightly depolarize your patched neuron to achieve spiking in a more reliable manner?
Good Luck!
Debanjan
You could try the theta burst pairing protocol. Here you would pair each (or some of the) synaptic stimulations during the TBS protocol with spikes artificially introduced by somatic depolarizations. Set the baseline somatic EPSP value to be around 4–5 mV, and do not change the stimulus intensity or the RMP throughout the experiment — even during the theta-burst pairing protocol. This is one of the most reliable and robust LTP protocols for SC synapses.
Keep the stimulus electrode close (40–50 µm sounds fine) to the neuron that you are recording to ensure robust and reliable responses even with low stimulus amplitude.
The protocol and the delay between stimulus and the action potential during the pairing protocol may be found in the following publications. You will note that you can modulate the number of pairings per burst during the protocol to alter the mechanisms and the strength of the LTP induced.
If you're blocking inhibition, ensure that you make a cut between the CA3 and the CA1 to avoid activating the CA3 recurrent collaterals through antidromic stimulation.
References:
http://science.sciencemag.org/content/275/5297/209.long
http://dx.doi.org/10.1038/nn1178
https://www.nature.com/articles/nn1568
http://www.jneurosci.org/content/26/47/12143.long
https://physoc.onlinelibrary.wiley.com/doi/abs/10.1113/jphysiol.2008.158816
https://www.sciencedirect.com/science/article/pii/S089662730700829X
https://www.nature.com/articles/nn2041
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