Thank you very much William M Connelly

Actually, we are doing calcium imaging with the OGB-1 dye. By adding the TTX, the calcium waves (synchrounous activity) get eliminated, but a single (non synchrounous neuronal calcium signals from neurons appears. This is done in organotypic cultures from rat cortex at 12 days in vitro.

why then the calcium waves is blocked by TTX whereas the single calcium transients stays?

Thank you again

The cortical organotypic neocortical cultures exhibit calcium waves in early development as in vivo. The waves that we see are spontaneous and not triggered. If we apply CNQX or APV or TTX (separately), these waves get eliminated.

We are trying to answer a reviewer question regarding our manuscript!

We developed a new method to load calcium dye with high effeciency. We could show in the manuscript  with this loading method that we can record calcium waves and block them with CNQX. The reviewer is concerened that may be with this loading method we are not able to detect action potential!!!

Unfortunately, we dont have in our lab an electrophysiology setup. Therefore, we thought to apply TTX and see if TTX can block this single calcium transients (Asynchrounous activity) that we see. Without any stimulation we due two types of activity with calcium dye

1- calcium waves (synchrounous and for all neuron)

2- single neuron calcium transients (Asynchrounous and not all neuron exhibit this transients). We believe that this second activity reppresents action potentials and we are trying to block it with TTX but we couldn`t.

Thank you very much! I will give it a try

They could also be trp channels, which can be activated by a variety of means, some are stretch mediated, via signaling cascades, etc. TTX won't block trp channel mediated calcium currents. There are a number of inhibitors specific to different families of trp channels.

Yes, I do agree also. I tried to block with cadmium or Nifedipine or  APV or  CNQX but is still calcium transients going on.

Mohammad,

It is also possible that 100nM TTX is not high enough to completely block Na+ APs. Check figure 6c,d in the paper: "Slow Recovery from Inactivation of Na1 Channels Underlies the Activity-Dependent Attenuation of Dendritic Action Potentials in Hippocampal CA1 Pyramidal Neurons. Costa M. Colbert, Jeffrey C. Magee, Dax A. Hoffman, and Daniel Johnston. J Neuroscience 1997."  Hope this helps.

I generally find that 500 nanomolar TTX is required to block all Na-dependent spikes in mouse cortical slices.  I often use 1 micromolar to be sure.  Perhaps it is as simple as that.

Dear Dr. Hamad:

The IC50 of TTX in dissociated neurons and in expression systems is around 3-5 nM. Typically, for slices the concentration of TTX should go up X100 times. If you are recording deep in the tissue, this amount may also be not enough to achieve a  complete block of Na+ currents, so you have to double the concentration. Keep in mind that the time to the onset of the effect is also much longer if low amounts of toxin is used.

Even after using 1 mkM TTX, you may see spikes, depending on the type of neurons you work with. In principal neurons of olfactory bulb and cerebellum, these are typically Ca2+ spikes generated in dendrites. They are slower but are spikes. In cortical neurons and basal ganglia, such spikes are rare but can also occur in the presence of TTX.   

Thank you all for the comments

To Dr. Saak V. Ovsepian

Do you know any citation claiming that "spikes in the neocortex are rare but can also occur in the presence of TTX".?

Dear Mohammad - these have been shown by many groups. Buzsaki was one of the first who showed them in the hippocampus in vivo about a decade of more ago. In the cortex these have also been reported - perhaps as a starting point you should check Greg Stuart's papers. Dendritic Ca2+ spikes are actually quite common in cortical neurons, but typically are shunted by K currents and fail to reach the soma. They become very robust in the presence of 4-AP.  Best wishes, Saak  

We typically use 500 nM TTX but find that remaining, are EPSC from occupation of AMPA receptors. This is why 10 uM CNQX really makes things quiet (Ca2+ levels drop). This is not using slices but cultured neurons.

How do you differentiate between spikes and mEPSCs?

I mean in terms of amplitude, rise time, decay time etc?