1) Increase potassium chloride concentration from 3 to 5 mM.

2) You wrote low Mg, best would be in your condition 0 Mg (Mg free ACSF)

3) if both of the above condition does not work then prepare transverse slices

This is an interesting question. I have used the no Mg preparation for several years. It works well for me so I do not add anything in addition. The other similar preparation I used before is a recipee with bicuculline. With regard to slicing directions, I have seen some studies in the literature that imply that the region of the hippocampus and the slicing direction do make a differernce with regard to seizure-like events. So I would try this.

Thanks Abdul Wahab. I will try increasing kcl in my next experiment. I prepare zero mag solution, but call it low mg, since there may be some mg remained in the tissue from normal acsf of holding chamber.

If i dont get longer sles, i will try transversal slices as you mentioned. Do you recommend 400 um width?

Thanks

For the rat most people use 400um width and for the mice most people use 350 or 400 um. I think you should try one of these for mice. But it should not be more than 400 since mice brain is smaller and you will get very few slices if you prepare more than 400.

For me 400 has worked best and in my lab every body is working with this thickness, so I will recommend this based on our experience.

You will probably get better preservation of the CA3-CA1 pathway with transverse sections. You can check the integrity of the pathway by placing a stimulating electrode in CA3 and recording in stratum radiatum of CA1---with healthy transverse sections (250-300um), you should easily elicit fEPSPs of 1 mV or more.

I tried coronal slicing with 400 micrometer thickness but I did my recording in a low Ca and high Mg condition which is totally different from yours ! I think using DNQX helped me a lot to get very clean recording! what do you record? mIPSCs?

Based on the comments I have got up to now, I will try doing these to get long SLEs (~ 10 sec):

1. Increasing potassium chloride concentration from 3 to 5 mM.

2. Using transverse slices.

3. Using bicuculline, or DNQX.

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Alexander,

A soon as trying transverse slice, I will update you about the result.

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Arash,

Thanks for your comments. I am recording field potentials. The dynamical aspects of SLEs are important for me. I investigate them from dynamical systems point of view. I am trying to use these data to tune my theoretical neural model.

You mentioned about "clean" records. Do you mean higher signal to noise ration by "clean"? I am recording remarkably low-noise events, but my problem is about lack of long-lasting seizure like events in my records.

yes I mean higher signal to noise ratio... how much is your Ca concentration in your choline solution?

Arash,

My No-Mg recipe (5 mM K) contains 2 mM CaCl_2.

Abdul Wahab,

Just double-checked my No Mg composition, I use 5 mM of KCl. Looks like that using transverse slice is the first thing that I should try soon.

Ehsan,

Have you checked the temperature? Maintaining a temperature of 34-35C is important to elicit ictal-like bursting in slices. Lower temperatures (

There are many factors that can influence the production of SLEs

Age is critical: the older the animal the more difficult it is

Slice thickness, the thicker the better (you need the max of connectivity), with mice you can go up to 500-600 um in adult

Perfusion speed, the faster the better, up to 8-10 ml/min

Temperature, must be physiological, around 35 (can be tricky to maintain at high perfusion speed without degassing)

Slice orientation. If you cur sagittal, use slices from dorsal hippocampus (parasagittal may work, but I have not checked).

Increasing K, and bicu may work, but I have experienced that too epilepetogenic conditions kill epilepsy

If you want to benchmark your preparation, just use 0 Ca ACSF, which invariably triggers SLEs (check J. Jefferys' papers). That way you are sure that everything works (slice orientation, hippo-Cx propagation etc).

Christophe，

I have learned a lots from your answer.

How to keep hippocampal slice acitivity in older mices like 6 weeks?

In adult slices, the best procedure is to perfuse animals with ice cold modified ACSF. There are many different ways.

Since performing dendritic recordings critically depends on slice quality, you can check the method in

http://www.nature.com/nprot/journal/v1/n3/full/nprot.2006.164.html

if you want good activity, it is better to use the dual flow chamber:

http://www.ncbi.nlm.nih.gov/pubmed/19200237

hope that helps

Hi Ehsan, in addition to what the others have said you may want to do your best to preserve connectivity between hippocampus and cortex - the best way to do this as demonstrated by Tor Stensola in the attached pic is to cut Horizontal slices. The attached pic shows how to this, ie what angles to slice at. I've found this slice prep to be really great for getting spontaneous activity that can propagate between the two regions, although I haven't tried getting ictal events using these slices.

I have however used organotypic hippocampal slice cultures and there it is much easier to get ictal events lasting quite long between 10 and 200 s. Interestingly, in these slices lower temperatures often result in longer seizures that occur less frequently, but higher temperatures have shorter seizures which occur more frequently.

I have read in the literature that SLEs transistion to short recurrent discharges of large amplitute (resembling interictal like activity but with greater occurence ). However i cannot observe that in my 0 Mg preparation as i stay in SLE phase. I am also using coronal slices and conduct the experiments in 34 deegrees celcius. Does anyone know why is this happening ?