I have experience with post-natal hippo and cortical neuronal cultures. I harvested the tissue and used papain to digest it. I then triturated with pipettes of different bore sizes and finally, used a Nycoprep gradient to isolate neurons. It is quite difficult and the yield is usually not excellent, but the more you practise, the more cells you will get. You can find the full protocol in our paper:

http://www.sciencedirect.com/science/article/pii/S0142961208010521

This is an adaption of the protocol used by Gregory Brewer and Bruce Wheeler mentioned earlier, so have a look at their publications as well.

Let me know if you have any more specific questions on the protocol.

It is definitely tough. I have tried cortical cultures and also was successful at it. I would say the success rate was around 70%. If you need to do whole cell patch clamping, I am sure you will have enough to try on. The most crucial point is the cells dont stick, so while changing media, you should be really careful. Otherwise, you should not have a problem. So I would suggest using different coat and try to check which one of all works.

Well this question is a little vague. Is there a particular neuron type that you are looking for? e.g. hippo, cort. dopam....etc.... Some have different protocols and some you can get from other sources. If you can give me that, I can give you a more defined answer, if that is ok with you?

We gave up trying to do that in adult and aged animals. We used different enzymes, different chelators, times, temperatures, antagonist cocktails, etc... The paper you posted is interesting but is still dependent on enzymes and trituration, which, inour hands yielded so few cells. Perhaps the glial "feeder" layer is the key?

Dr. Gary Brewer has several papers using special techniques and propriatory media for culturing aged neurons. When patch clamping NMDA currents (I think), however, the amplitude of the currents was very small (like ~30 pA) and frankly abnormal looking (normal in aged neuron easily reaches 2-3 nA).

If you can switch to single channel recordings, the "zipper" preparation (originally described by Rich Gray in Dan Johnston's lab) is much better and allows for acute dissociation of neurons with beautiful arborization. We have used this technique on many papers for analyses of aging-related changes in hippocampal electrophysiology.

Also for another acute dissociation prep, look up Murchison and Griffith or Alexej Verkhratsky papers.

Good luck and let us know if you succeed!

What age are you looking for and why?

Which region of brain do you want to make culture?

I have experience with post-natal hippo and cortical neuronal cultures. I harvested the tissue and used papain to digest it. I then triturated with pipettes of different bore sizes and finally, used a Nycoprep gradient to isolate neurons. It is quite difficult and the yield is usually not excellent, but the more you practise, the more cells you will get. You can find the full protocol in our paper:

http://www.sciencedirect.com/science/article/pii/S0142961208010521

This is an adaption of the protocol used by Gregory Brewer and Bruce Wheeler mentioned earlier, so have a look at their publications as well.

Let me know if you have any more specific questions on the protocol.

Yep, I totally agree: culturing neurons from postnatal and adult brains is definitely tough. I followed the protocol from Brewer & Torricelli in the past (see attachment) with papain, Neurobasal A, a homemade Hibernate A-like medium (the original from BrainBits is difficult to get out of the States!), etc. but still had problems, both for hippocampal and septal postnatal cultures. Dissociation is a key step, it seems: neurons are too differentiated at these stages for undergoing all the culture procedure without suffering major harm...

The purpose I had was not electrophysiological recordings, though, so I cannot discard that some protocol you might find is good enough for that - try some of the references suggested above, but make sure your culture is healthy enough before doing any experiment with it...

Good luck and let us know how it's going!

Did you know of this book?

Could be useful

http://books.google.com.co/books/about/Culture_of_Animal_Cells.html?id=Rq96QgAACAAJ&redir_esc=y

In the adult brain, the main obstacles will be the denser extracellular matrix (ie: requires optimization of the digestion conditions) and the lower tolerance to anoxia. Bubbling O2/CO2 during dissection is impractical in term of sterility but reducing sodium concentration and adding glutamatergic antagonists may help, as suggested before. You may find useful tip in the literature where acute adult slices are prepared for patch-clamp recording. We had some marked improvement of survival for adult cerebellum by adding polyanionic polysaccharide (Dextran) to dissection/digestion media.

I hope this helps

Thanks so much for all of your comments. I will look over the references and see. We want to try cortical, hippocampal and perhaps cerebellar cells.

We should probably talk. There are issues that none of the others have addressed. I did Patch Clamp with neocortical NMDA cells that I derived from Mouse fetus thru adult Rat at NIH. My mistake was to show that there were flaws in the existing protocol that changed the initial result from the research. I developed a protocol that did great with a high viability concentration and demonstrated (or substantiated) physiological events shown in human clinical trials. I showed that, for one, Enzymes created second-messenger involvement within the cells. i tested many enzymes and purities and found the same. I also found issues with the Carbogen supplied to the entire East coast of the US. It turned out that the Co2 contained a contaminant (cyanide) which even though the supplier tried to tell me was only so many PPB, and should not do harm to my cultures. I told them that it was enough to kill my neuronal cultures and when the Carbogen was cleared of it, my cells thrived. I don't know if they fully removed the contaminant because I was shut down just as I started back up and again the supplier still felt that it would do no harm. I published abstracts at Society For Neuroscience meetings and was subjected to slight condemnation from established Emeritus researchers, but None could disprove or give cause to reject my findings (instead my time was ended and money dried up). I still find it funny that people still use that enzyme protocol as a standard. However, when anyone publishes having used an enzyme protocol for disassociation, I take their results with a grain of salt with possible flaws.

Plz check this paper fpr details

Fatima-Shad, K. and Barry, P.H. (1998). Morphological and electrical characteristics of post-natal hippocampal neurons in culture: the presence of bicuculline- and strychnine-sensitive IPSPs. Tissue & Cell, 30 (2) 236-250.

Plz check this paper

Thank you

Hi, I've just started trying to acutely dissociate pontine (Locus Coeruleus) and midbrain (Substantia Nigra pars compacta) neurons from jouvenile rats and mice. Could anyone please recommend digestion times? I'm currently punching out the regions of interest and digesting them in papain for 30 min at 34 degrees C.

Secondly, how much should I titruate these tissue punches following enzymatic digestion? Should I be aiming to break up the tissue pieces entirely? Or is gentle titruation leaving the tissue punches intact enough to knock a few viable neurons free?

Cheers, Andrew