Hi Dina,

I think it's most practical to stick with a bipolar stimulating electrode, not sure what other configurations you were considering - like a concentric (single-point) bipolar for example? The reason being that the synaptic connections are not going to be organized in such a way as they would be in brain slice - certainly not like in laminar structures like hippocampus or cerebellum. Actually for cultures, the bipolar electrodes can be much more widely spaced than ones typically used for brain slice. Essentially you can have the poles straddle both edges of the coverslip, if you wanted to maximize chance of stimulating inputs to the cell you're recording from.

As for the stimulator, I'm not sure if you're referring to the output (V vs I), but I've used ones that are easily switched from constant voltage output to constant current output - I typically use constant current output. Haven't tried constant voltage output when stimulating cultures, but I know in slices, both output modes produce effective stimulation.

Best,

Hi

I agree with Haider's answer. Bipolar stimulation is far more effective and focal, and the use of 'side by side' electrodes would be more effective in cultures than concentric. Certainly this will be less likely to 'fry' your cultures as long as you keep the current (constant current mode is better) to appropriate levels. Any gas bubbles and you are too high!

Biphasic stimulation tends to prevent polarisation of your electrodes and prep with time, and eliminates the need to periodically reverse the electrode current. Just be mindful of which electrode you call the 'positive' electrode, being the first electrode to go positive in a biphasic pulse with reference to the other electrode. Reverse the polarity to determine which direction is best - sometimes this makes a difference in in vitro preps. 

Kind regards

John Reynolds

Thanks so much for your suggestions! I have a few follow up questions.

If my bipolar electrodes straddle the coverslip what would be the chances that I would get antidromic AP as opposed to orthodromic? Does it really depend on how far away from the cell I'm patching or internal patch solution or something I will have to play with to figure out the current to use?

John- I am bubbling my aCSF solution so is that going to be a problem since there will be bubbles around? Also are you saying biphasic would be a better choice with bipolar electrodes straddling the coverslip? Also, maybe Im not understanding the electrical circuitry of this well but why does the (bipolar) electrode get polarized enough to have to reverse the electrode current periodically? And how does that impact patch electrode recordings?

Can you suggest a stimulator manufacture I know digitimer has all 3 kinds but are there other ones you would recommend over digitimer?

Hi Dina

We use the digitimer for the stim isolator and for the unit that drives the stim waveform and it works well. As long as you are getting a nice square pulse and the stimulator does not generate any residual current or noise when you are not stimulating most brands are fine (this is more a problem with biphasic stimulation usually since the output stage has to be 'balanced' to produce no residual current when your drive waveform is at zero).   I am not really used to doing this type of stimulation in cultures (more experienced with slices) so I will leave others to comment on antidromics  - I guess you go from edge of slice to edge of slice to maximise chance of depolarising neuronal elements so maybe that is better for cultures.

If you pass the same current in the same direction onto two electrodes in bipolar configuration enough times it is theoretically possible to build up a charge between them. Not so good for long-term plasticity studies but maybe this is not an issue for you. 

I was talking about bubbles coming off the tip of the stimulating electrode with very high currents (you shouldn't see it really in usual practice) not bubbles from ACSF movement.  

Good luck!

Hi Dina,

I guess I didn't pay close attention to what it is exactly you were looking to record from your preparation, and assumed that you're interested in looking at synaptically evoked currents (not APs). My answer was basically saying that if you're already equipped with the hardware to carry out brain stimulation in brain slice, then you don't need anything any more specialized for starting experiments with cultures.

For brain slice work, I use fine and closely spaced electrode tips manufactured by FHC; since these are precious for slice work, I don't use them for cultures grown on coverslips because it's easy to damage the tips when lowering the stimulating electrode to the surface of the coverslip (it's hard to resolve a monolayer of cells with coarse objective). So the stimulating electrodes we use for cultures are home-made (and rather crude!) - basically two silver/silver chloride wires held close together using epoxy and threaded through plastic and/or glass pipettes.

Now, about the ortho/anti-dromic business - I am under the understanding that that does not apply in culture...mind you, I'm also under the understanding that it doesn't really apply in slice experiments with evoked stimulation either, but if others coming across this thread think otherwise, I'd love to learn of their thoughts on this...I've already learned something new here from John about biphasic stimulation, so it would be good to learn more...

Back to electrical stimulation in cultures...basically, the axons in dispersed neuron cultures are going to be traversing every which way, so when evoking excitatory transmission, that most often leads to very broad (and very messy looking!) synaptic currents due to recurrent excitation (hence why most people stick with mEPSC when examining excitatory transmission in culture). Now, I've seen in the literature examples where the evoked excitatory currents look somewhat like they do in slices; perhaps if that's what you're looking to accomplish, then you can stick with finer/closely spaced electrodes, and of course using the lowest possible I/V output settings. But chances are you won't always wind up with the electrode placed in the "right" spot to stimulate the synapses of the cell that you're recording from. On the flip side, if you were ever interested in looking at evoked inhibitory transmission, then the widely spaced electrodes are great for that, since the recurrent excitation is not an issue there, and you can stimulate at high frequencies.

Hope this helps,

For brain slice I have also used the bipolar concentric electrode from FHC which work great in the hippocampus. There definitely is a difference between antidromic and orthodromic since with antidromic you dont have a AP threshold. You see instantaneous spike so its a good measure if you want to look at accommodation but not AP properties. So I worry about this but I'm going to try this same kind of stimulation with my voltage input and same electrode on these cultures tomorrow...I hope i dont break the electrode going too close to the glass. That is something really important to consider though because the chances of ruining it are very high.

Thanks so much for your advice and I'll let you know how that goes!

Dina

If you break the glass then chances of frying are really high :-). I have been through this. I with Prof. Reynold's suggestion.  

That's useful information to know that ortho/anti-stimulation makes a difference in terms of how recorded APs appear, although don't think I understand why that's the case? At any rate, best of luck with your experiments!