Hi,

I've never record antidromic APs, but used to record synaptic currents.

So I thought I would respond with something.

First, I simply think that stimulus artifact is not the problem in itself. You can minimize the artifacts by several ways, but a certain size of artifact is unavoidable.

Your problem is rather that you cannot get APs and EPSPs, I think...

Additional information is needed to go on.

>I am currently doing brain slice physiology

1) What is the type of neuron you are recording?

>I was only able to get transient artifacts (see attached image).

2) What is the stimulus frequency and duration in the attached? Assuming that the basal noise is in 50Hz, the artifacts seem to be too broad. By the way, as Dr. Galvan pointed above, you may want to reduce the hum noise more...

>I was able to elicit some EPSPs, but was still getting an artifact at the very beginning of the EPSP

3) How far is the electrode from the cell? Keep the electrode away from the recording cell as possible. Naturally, artifacts can be smaller and the synaptic latency can be longer as you stimulate further from the cell. When the stimulation is too close and too strong, presynaptic terminals are stimulated directly and action potential-independent EPSPs would be evoked. If you have to bring the electrode very close, discard the cell and let's move on to the next cell/slice.

4) How thick is the slice? Can you see spotaneous EPSPs? If you can see them enough, it means that neurons are healthy but, unfortunately, axons are discontinuous. It might be a good idea to review slice preparation.

>I've tried different stimulating electrode types, reversing the polarity of the stimulus, and changing the stimulus amplitude, but to no avail.

5) What is the type of the stimulus electrode? (monopolar or bipolar?, tungsten wire or glass pipette?) Artifacts can be broad depending on the type of the electrode.

6) Have you tried to use voltage stimulation? Artifacts evoked by current can be broader than those by voltage, because of the capacitance and resistance of electrodes.

Hope it will help you.

Hello,

Thanks for the tips.  I'll try to answer all your questions in an organized way to make the discussion easiest for you, since you are so kind as to help me out.

Grounding and equipment:  I ground all equipment in a Faraday cage.  For a grounding electrode, I use a Ag/AgCl coated wire that is submerged in the bath (this wire is thicker relative to the wire I use for recording electrodes).  For recording electrodes, I also use a Ag/AgCl coated wire.  I typically rechloride wires every week or every other week.  All other equipment is sufficiently grounded, and I tend to check grounding on a regular basis using the trick of grounding myself and touching different equipment (as Dr. Galvan noted).  I haven't detected any sources of noise thus far, but perhaps I'm missing something.  I also feel it might be a good idea to check if my Faraday cage is properly grounded and is shielding the system sufficiently.  I'll investigate more to see what I can do in terms of any noise.

Neuron type:  I'm recording from mouse CA1 pyramidal neurons.

Stimulus frequency and duration:  Frequency of stimulation differs between protocols.  In the trace attached, the protocol is set to stimulate a train of 0.5 ms pulses at 50 Hz.  I've also attached another trace to include the time parameter.  The two traces attached are from a 10 Hz protocol.  One trace incorporates the whole protocol while the other is substantially zoomed in to show one of the artifacts completely.  Additionally, I included the x-axis on these traces to give that dimension as well.

Distance from the cell:  The electrode is relatively far from the cell.  When trying to stimulate antidromic action potentials, I place the electrode near the subiculum to try and ensure that I stimulate the axon of the CA1 pyramidal cell and ensure I'm not too close to my recording electrode.  Similarly, with EPSPs, I try to place the electrode closer to CA3 in stratum radiatum for the same reason, but with Schaffer collaterals rather than CA1 pyramidal cell axons.

Slicing:  I don't believe slicing to be a problem, though I may be wrong.  I don't really observe spontaneous EPSPs very often.  Occasionally 1 or 2 may pop up while I'm recording, but if I see probably more than 3 or 4 in one trace I cut that cell loose and find another.

Stimulus Electrode Type:  The traces here are from a broken tip glass pipette made with theta glass and filled with aCSF, and using (I believe) tungsten wire.  I also tried a monopolar electrode and got roughly the same result.

Voltage Stimulation:  My PI has suggested this, but I haven't tried it quite yet. I will likely try this soon to see the difference.

Thanks again for the suggestions!  Your help is much appreciated!

I recommend to try out what has been suggested by others but I am sure if these noise are from your phone; because the noise from phone appears very different than shown in the picture.  However other possibilities cannot be eliminated! Not sure what type of chamber you are using but sometime I encounter similar things due to inappropriate liquid flow via inappropriate reference electrode (e.g. not coated properly or debris in chamber etc.). Thank you!!

You don't appear to have a "problem" to me. You will always get a stimulus artifact at sufficiently high stimulus intensities. You have an electrode in a conductive medium and you are applying a large electric field to that medium: this will create a voltage.

If I were you, I would be more concerned about your not seeing spontaneous any EPSPs and not being able to regularly recruit EPSPs when you stimulate stratum radiatum. There should be noticeable spontaneous EPSPs in a recording from CA1. Similarly, you should practically always be able to recruit EPSPs when performing field stimulation.

It looks like your field stimulation pulse is 1 millisecond long. This is a bit longer than most people would use. Typical field stimulation is 100-500 microseconds. I tend to stick with 200 microseconds.

 Sorry for late response. This is a quick note to tell you.

Your grounding seems appropriate. It may be better by rearrangement of equipments.

I said that your artifacts are too broad, but it might be not, as far as I see the new attached trace. Sorry...:(

The stimulating positions are probably OK.

About spontaneous EPSPs, I totally agree with Dr. Connelly. You will see many spontaneous EPSPs (not spontaneous Na+ spikes of the recording cell), if you don't add synaptic blockers to ACSF.

And if you can't see spontaneous EPSPs enough, it may be related to the other problem about antidromic APs.

hello everyone

I have a quick additional question to this conversation

I'm also doing evoked EPSC recording in 250µm hippocampal slice, I'm stimulating using a bipolar tungsten electrode from npi (500µs)

the problem is that I can recruit EPSC but the amplitude will decrease gradually and rapidly so I can't have a stable baseline

I'm also recording spontaneous EPSC I can see a lot of them at the start of the exp but once I add GABA(A) and GABA(B) blockers most of those disappears

Any insight to help me troubleshoot the problem, I'd greatly appreciate it