Dear Kueh, I have recorded from mouse and rat cultures at different time points after plating and there is certainly a lot of maturational changes also after DIV14. We also use neurobasal media and B27 supplement. I guess the question is what kind of matuartional stage you want to investigate. There are usually few spines at DIV14 and many properties resemble those of immature neurons in the brain. Although also not mature, we prefer to analyse neurons at approximately DIV21. DIV3 is of course even more immature and I there is little if at all communication with other neurons in the dish. Best, Jakob

Dear Sindy,

I've measured several active membrane properties from mouse and rat primary cultures starting from 6 DIV as far as 29 DIV using pretty much the same culture conditions you reported (Note: what about your coating?).

What I can suggest you, since you mention firing properties among your interests, is to wait at least 18 DIV to make good measurements of Current Clamp spontaneous and evoked firing.

If you need further help, let's say unrelated neuron development, to design and tune your patch clamp measurements in culture please drop me a line, I'll be glad to give you some hints about intra/extra cellular solutions which can help you to isolate specific parameters during your experimental sessions.

Good luck,

Sebastiano

Dear Sindy

I am doing also same kind of experiments. Normally action potential should be recorded at or after DIV 14. Neurons after 10 day in culture show mature action potential stablish its RMP more negative and show repetitive firing. some potassium channels change their pattern and expression during maturation of neurons.

I usually get repetitive firing from 8th day of culture. this type of early firing stimulate lower current input and diminished at more current injected.

Regards

Dear Sindy,

we made some preliminary tests in the first three days and in voltage clamp configuration we found only Outward currents but never Inward current for depolarizing steps.

Hi everyone, Thanks for the information. I am not really a cell culture person... when you said DIV 14, does that mean day 14 in culture... apologies for the otherwise naive question... :) We are mainly a protein/ electrophysiology lab... I am doing the culture for one of my colleagues as I was apparently the best person there, having some background in tissue/cell cultures... We will try recording at Day 21... THANKS!

My colleagues is interesting in the spike/action potential pattern in culture, compared to what is our normal prep (slices). He is also looking at back and forward propagation from the dendrites etc

And to answer Sebastian's question: I use Poly-D-Lysine for coating.

Will post what the outcome is ...

Dear Sindy,

Yes, DIV14 is day in vitro 14. DIV21 sounds much better than DIV3 or DIV14 if you are intersted in action potential pattern. Best, Jakob

Cheers Jakob ... :) Anyway, has anyone had any luck culturing adult mice neurons... I am also interested in making an organotypic cerebellar prep from 6-8wks old brain but haven't had much luck keeping them alive past 4-5days.

Since they are postnatal neurons, you should use neurobasal-A media with B27 supplement, and what I have found that the neurons began to fire action potentials around day 7.

And bFGF should be in the medium from day one.

Sindy, when you say you add "glut" to the medium, is that glutamate or glutamine?

Glutamate should, of course, should be left out.

We found opposite results from those you described in embryonic hippocampal cultures across 7- 30 DIV. See this paper: http://www.ncbi.nlm.nih.gov/pubmed/16914613

We used both current clamp and voltage clamp recordings. As cells mature, connections increase as do action potentials.

Hi Jonathan... L-glutamine.

Hi Sindy,

I've just come across your question, and it may be too late to help, but the following may help others looking for similar information.

I have been testing function of cultured dissociated hippocampal, cortical, and DRG neurons in culture with patch clamp every 5 days in culture till 25 days, then at 50 days in culture. These were all from late embryonic stage rats. I have found that whilst strong action potentials occur in hippocampal neurons by about 10 to 15 days in culture, they really require 20 to 25 days in culture to reach functional maturity to match recordings seen at 50 days in culture. This was also the case for cortical cells, although that data has not been published.

Both cortical and hippocampal cells were cultured in neurobasal medium with BOTH B27 and N2 supplement, WITHOUT L-glutamine. This is an odd combination, but appeared to give better function through long-term cultures, possibly by avoiding breakdown products of L-glutamine.

The attached paper shows the time course of activity in these cells in culture, I hope this helps.

Kind regards,

Justin.

Article Neuronal Electrophysiological Function and Control of Neurit...

Dear Abdullah, neurons spontaneously fire at some rate essentially due to membrane potential fluctuation that can bring them closer to firing threshold and their channel composition. Moreover, glutammate in culture bath also increases firing. Cultured neuron, then, also form synapses and with increasing div, their number and functionality increase. 

I do not include glutammate in culture bath, I suggest removing it.