Hi there,

my first time answering here but I'll try to help.

In A, yes that seems to be your fiber volley. In B the small peak right after your stimulus artifact seems to be an upward peak of your fiber volley. I don't see these myself as I work with low current stimulation, but it reminds me of the figure 3 in supplement of the paper "proBDNF negatively regulates neuronal remodeling, synaptic transmission, and synaptic plasticity in hippocampus".

The other peaks (in B, but most prominently in C) look like circuit activity. This is more prominent in the presence of biccuculine because inhibition is lifted.

And lastly yes, biccuculine is blocking GABA(A) receptors to diminish inhibition and ensure you are recording excitatory responses.

Hi,

I'm totally in agreement with Julio. All these peaks that you see after bicuculline perfusion are called "population spikes" and they are normal since bicuculline blocking GABAA receptors disynhibits the circuit and neurons start firing all togethere. I do not know which kind of study are you performing, in case of LTP experiments at the SC-CA1 synapse you can avoid bicuculline perfusion. On the other hand you have to block GABAA transmission if you want to induce LTP in dentate gyrus. Anyway, If you search for population spike on google you will find a huge amount of information about this phenomenon.

Have a nice day

Luca

I agree with Julio also. I just wanted to add, you'll probably get nicer looking recordings if you amplify you signals a bit more. It looks to me like you're right down in the digitizer noise.

Hi Hong

I agree with what was written previously and you spotted correctly the fiber volley and the fEPSP. On the recordings you presented, what is striking between your images and the one from the publication (in D) is that the ratio in the amplitude between the FV and the fEPSP is much smaller for you recordings. It is a sign that your slices might be unhealthy, which make them also more prone to produce population spike even in the absence of GABAA blockers. Another problem I see is that you stimulus artifact should not become biphasic (in pic B) when you increase the stimulation intensity. You might have to fix something with your stimulation electrode.

Thanks everyone for your help. Over the past couple weeks, I seem to have found the problem: unhealthy slices. The healthier the slice, the lower ratio between presynaptic fiber volley and fEPSP amplitude. The upper limit of the fEPSP amplitude will also increase, so I can use lower stimulation intensities to generate traces without population spikes, but at amplitudes similar to the publication images I attached. I definitely appreciate all the input.

William, when you say amplify the signal more, do you mean increase the stimulation intensity, or some setting on the amplifier? Is there an optimal gain I should be using? For now, I use gain of 1 and signal is filtered at 10 kHz.

Sam. Yes, there’s definitely something weird with that biphasic event going on… Sometimes it’s there, sometimes it’s not. Will have to do more experiments to see if I keep getting them in healthier slices.

Hi Hong,

I mean some setting on the amplifier. A gain of 1 is definetly not optimal. If 10x or 100x gain is available, I would use it.

Classically, you set the gain so that the signal you a recording nearly "fills up" the digitizer. What this means it that a typical digitizier will want signals between -5/+5V or -10/+10V. So you want to amplifier you ~10mV signal so that it ~5-10V...

These days, because digitizers are better than they used to be, you don't need to amplify so much, but I would definitely look for an increased amplifiation: your signals will look cleaner.

Hi Hong,

I do the same thing and at the beginning I wondered the same. What you see in your first image is indeed the fiber volley as everybody pointed out. No need for bicuculline as well, I do the same kind of recordings in the CA1 and I don't need bicuculline.

What is the age of the mice you use? Have you tried the recovery method with NMDG (instead of Na+ in your aCSF), to try to increase the viability and the fEPSP amplitude in your slices? I truly recommend it!

Good luck!

Vincenzo Mastrolia Hi Vincenzo, would you please tell me the details of the NMDG recovery method for fEPSP recording with adult mice? Are you following the original NMDG method (Article Acute Brain Slice Methods for Adult and Aging Animals: Appli...

I can get nice whole cell recordings with 12-month-old mice using NMDG recovery method (the optimized one published in 2018, https://www.jove.com/video/53825/preparation-acute-brain-slices-using-an-optimized-n-methyl-d ).

Also, I can get nice fEPSP (about 2-3 mV at most) and LTP in SC pathway with 2-3 month-old mice using sucrose based cutting solution.

However, the fEPSP is only about 0.3 mV when I use 6-month-old mice and optimized NMDG recovery method. The slice is 350um by the way. I am thinking whether I should not do the transcardial perfusion as NMDG left in the brain slices could potentially shut down all the neuron activities.

Looking forward to your reply.

Dear Danlei (Flora) Bi, could you please tell, did you solve the problem w/ low fEPSP from 6 months mice? If so, how? And did you eventually stop using NMDG for transcardial perfusion? Sorry for the late request, but I'm currently struggling with proper LTP induction in 24-month mice, not exactly knowing what's wrong(

Hi Denis Ashrapov and Danlei (Flora) Bi , apologies for the late reply! It has been a while, I know. It is true that NMDG can be toxic for neuronal activity if left for too long. However, I found two different solutions both valid to ensure to have the benefits of NMDG but without consequences:

1) Use NMDG for cardiac perfusion, cutting and recovery at 32 degrees C but being extremely fast, in order to put all the slices into recovery within 10 minutes from decapitation

2) Use a low Ca2+/high Mg2+/high HEPES aCSF for cardiac perfusion and slicing and leaving the slices in NMDG only for the recovery at 32 degrees C. Ting lab (the same as for the jove method) has shown that NMDG makes substantial difference during the recovery but it is not essential during the other steps.

Also, consider that the older you use the mice, the more impactful is the temperature. For very old mice, cutting with a slushy aCSF and putting the vibratome's cutting reservoir well in advance in the freezer can make a huge difference in term of neuronal survival.

Hope this helps! If not, don't hesitate to contact me in private.

Cheers,

Vincenzo