Hi,

I never found problems related to the extracellular matrix while patching. How old are your animals? Usually with old animals the visibility deep in the slice is significantly reduced and this may affect your sealing. Slices obtained from old animals are often of poor quality. If you see a clear dimple on the membrane the chances to get a seal are very high. Check some methods for improving your slice quality and visibility.

http://www.brainslicemethods.com/

Thanks for the Davie paper, hadn't come across it before, and I actually just made up a batch of the NMDG recovery solution (the lovely lifesaver)!

These animals are wild-caught amphibians, so their age is unknown but potentially as old as ~15 years, a figure which might the blood of those working with juvenile mice run cold. The slices and the cells are typically quite healthy, just obscured by thick matrix - I can see clear dimpling, but in between the dimpled cell and my pipette, alas, lies matrix. 

Keeping a high pressure in the pipette will help and gently moving the pipette around on top of the cell for  sometime and you will  find a clean spot and need to be patient.

Good luck!

This paper also has some worthwhile discussion of alternative methods to keep your tissue healthy, similar to NMDG-replacement - http://www.ncbi.nlm.nih.gov/pubmed/21084688 and I think making sure your tissue is as healthy as possible is probably your best bet, giving yourself as many options as possible when trying to patch.

You could also consider using a second electrode setup for pressure ejection to locally apply enzymes to dissolve the ECM. As you said, it will make the prep less physiologically relevant, but restricting the modification to your cell will at least leave the local network intact.

We had a similar problem with chick brainstem - it seemed to be intermittent but we could never figure out what actually caused it.  We did try several enzymes (as suggested by Steven above; its something I have a bit of expertise in) but without any clear success and I too would love to hear of something that works.  The problem is that the strong enzymes (trypsin, papain, etc) break cell connections.  I think more work is needed on the CNS extracellular matrix to identify the right enzymatic approach.

One thing we did not try that you might think about is to make organotypic slice cultures.  There is a good chance that the extracellular matrix will clear on its own accord - but of course there are many other changes that will occur in time.

I hope someone has the magic bullet!  [Jonny: thanks for raising this question.]

Appreciate the replies y'all. 

Agreed that tissue health is critical, Steven. I've found that keeping a low 'time from decapitation' seems to best guarantee that (in addition to the stated prep techniques). This not only leaves more cells alive from which to choose, but makes them better able to withstand the needed disruption of the matrix. 

Shockingly, this week I've had very good luck sending in a first pipette and *actually attempting a patch* on a cell of interest, then pulling that pipette back such that the cell is very slightly stretched and applying the short burst of suction typical of breakthrough at the same time as rapidly pulling the pipette back with the micromanipulator. This was initially done as frustration after a failed GOhm seal, but it seemed to rupture both the extracellular matrix and peri-neuronal net without killing the cell allowing another pipette a successful patch. Extremely resilient little dudes, these cells are.

I've also managed to break the matrix by lowering a relatively large (3-4MOhm) pipette that is unfilled into the bath and allowed it to passively attach to the matrix as it front-fills. An alteration between light suction and slow, low amplitude, but long repeated movement picks up a lot of the matrix - rinse and repeat until the cell is clean. For a particularly stubborn matrix I pulled a pipette to a point and jousted diagonally (it helps if you're playing medieval English music and fretting about the amount to which your glass is doped with chivalry) into the matrix until I made a hole and then proceeded with the above.

Certainly going to try enzymes in the future, but deadlines approach! I'd also love to try organotypic slice culture, but the same applies.

^An addendum to the 'time from decapitation' comment - This week I've found that even with a relatively brutal "severe brain damage guaranteed" dissection, as long as the time was short I found healthy cells.

Dear Jonny,

First, I wanted to let you know how much I have enjoyed reading your posts.  You have a great humorous writing style that captures the frustration we all have at the bench.  Now I will always view micropipette placement as jousting.

I am not an electrophysiologist (I just do enough to attempt in vivo electroporation and helped map visual responses with fEPSPs), but there have been several in my mentor's lab that have tried whole cell patching on the retinorecipient neurons at the bottom of the SFGS in the goldfish tectum.  They are pretty small (~ 10 um), so they finally went for a perforated patch setup that used gramacidine in the pipette (evidently nystatin didn't work well).  So I was following this thread to see what others had found that might work in our lab.

My question is for you and Elise.  Since mammalian perineuronal net is usually rich in CSPG, has anyone tried chondroitinase ABC to clear out the ECM?  I didn't have time to do an exhaustive Pub Med search, but no one seems to have mentioned using it.  Unfortunately, CSPG is involved in modulating neuroplasticity, so I am not sure if it would be a good idea.  But then, all the other enzymes have effects, too.  Just curious.

Jonny, I am glad something finally worked!

Jill