I am using MEA device to perform extracellular recordings in cortical primary neurons. My cultures show synchronized bursting activity, however as a negative control for some of my experiments I need to decrease that activity.
Does someone know which kind of compounds can I use to decrease the synchronized bursting activity in a culture?
Thanks for the help!
depending on what timescale and level you want to act on:
something that blocks excitatory neurotransmission (like cnqx 40uM) will decrease bursting frequency by reducing firing rates, kynurenic acid can all but eliminate it
something that blocks spiking (like TTX 1uM) will also obviously eliminate bursting
muscimol will decrease bursting by hyperpolarizing a lot of neurons
or do you mean you need to decrease synchrony only?
Hi Rita,
In addition to Alberto's suggestions you can try to halve your external K+ ion concentration to 1.5 to 2 mM.
best wishes, Refik
Thanks for the tips.
Alberto, answering your question I would like decrease only synchrony.
Dear Rita,
since discoveries from different groups (ex. from B. Barres), it is clear that glial cells play a key role amplifying a spontaneous activity of neurons in vivo, in vitro brain slices and in co-culture. So, spontaneous activity that you see can be due to it. I attached some PDFs (Ullian et al., (2001; Sloan& Barres 2014).
IF Yes, the glial cells are in your hands, then Refik suggestion to reduce [K] is absolutely correct for two reasons:
1. lowering external [K] you hyperpolarize neurons to set their membrane potential much below threshold when they spontaneously fire spikes, HOWEVER this is rather mild treatment since neurons do not sense [K] below 6-8 mM (Kuffler et al., 1966; Orkand et al., 1966).
2.-however, lowering [K] you MOSTLY hyperpolarize K-sensitive glial cells (if you use neuronal-glial co-culture), thus you will (i) reduce a release of excitatory transmitters from glia (such as glutamate (Parpura et al., 1994)) and (ii) by amplifying glial glutamate transporter (due to hyperpolarization), the cleaning of glutamate (and other neuroactive substances) from extracellular space will be better. This will reduce spontaneous neuronal firing.
3. You can also use IEM-1460 (GluA-2-lacking APMAR blocker) to quench excitation and therefore calcium overload in cultured neurons to rescue the neurons from overfiring and even from apoptosis as well (Abushik et al., 2013).
Also, if TTX is not a way for you (hard to wash it out), you may use any blockers of NMDA, AMPA receptors to avoid spontaneous firing because you will block excitatory inputs in neurons.
4. Epileptiform activity was found in primary culture of cortical neurons and can appear spontaneously as you see in your experiments. Since such firing can be triggered by (i) the withdrawal of magnesium block of NMDA receptor channels or (ii) by shutting down GABA-ergic inhibition, you can increase either magnesium concentration from 2 mM to 4 mM in perfusion or/and you can add 1-5 micromoles of GABAa blocker bicucullin.
5. In all such cases spontaneous activity depends on intracellular calcium oscillations and you can depress it by nanomolar ouabain (Sibarov et al., 2014).
This is one of the best approaches using low doses of ouabain to amplify Na/K-pump and Na/Ca-exchanger that results in dramatic reduction of calcium overload in neurons and their excitation (Abushik et al., 2013).
Indeed, ouabain (a glycoside using in cardiac therapy, etc.) has two effects:-in high doses (millimolar or sub-millimolar concentrations) it blocks Na-pump and causes cell death (Akimova et al., 2015), while in low doses (nanomolar) it enhance the pump and robustly increases cell survival and reduces excitotoxic stress (Krivoi et al., 2004; Sibarov et al., 2014).
Interestingly, ouabain, as an endogenous digitalis like factor, also called old endogenous cardiac steroid, is present normally in the body at low doses. (Simonini et al., 2015).
Good Luck! Very cordially, Serguei.
Dear Serguei,
Thank you very much for all the tips! Indeed I also think that the best option for my experiments is the nM concentrations of ouabain.
Cheers,Rita
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