How to to make a movie about the life of the neurons

A Butcher, a Baker, a Candlestick Maker

by Anton Ivanov, PhD

One day you decided to make a movie about the life of the neurons in the hard
condition of the culture. So you plane to do a time-lapse recording. Really, it is a
good idea. The movie about neuronal existence is a very amazing show. It gives a lot
of pleasure!!!

What about scientific results? It is more difficult, but a pure
pleasure obtained during experimentation is itself a good result. Before caring out
time-lapse recording you need resolve some amount (roughly one hundred) of the
technical problems. One of these is the solution to perfuse you culture during
recording. I wrote "to perfuse" because if you plane a recording of few hours you
should continuously refresh the external solution to avoid a changes in the solution
osmolarity.

The ideal external solution for this purpose is the same that you use to
incubate your cultures, so called Minimum Essential Medium. The ideality of this
solution is only theoretical. First, MEM contains NaHCO3, so to keep pH at
physiological level you need have exactly 5% of CO2 in the MEM and in the
environment arounding the recording chamber with the culture. This requires some
supplementary equipment. Second, the MEM contains phenol red that is auto
fluorescent and makes impossible the recording of the fluorescent cells.

There are some other reasons that make difficult (i.e. impossible) the using of MEM for
time-lapse. Such difficulties brought me to the decision to cook myself an
extracellular solution for time-lapse. I refused to use CO2. In fact, the neurones
(cortical or hippocampal from embryonic or newborn rats) need CO2 at hight (5%)
concentration only during first four days of their development in the dissociated
cultures (Brewer and Price 1996). So, if you work with more adult culture the
ambient concentration of CO2 is sufficient. As pH buffer I have chosen a HEPES/HEPES
Na. This buffer is stable at temperature from 25° up to 37°, i.e. pH remains within
physiological range (7.2-7.4). In all others aspects I tried to mimic the hibernate
solution proposed by Brewer and Prince that I yet cited above.

I have used the inorganic salts that I normally use for extracellular solutions for patch-clamp
recording (mM): 140 NaCl, 2.5 KCl, 2 CaCl, 2 MgCl, 20 D-glucose, 10 HEPES/HEPES Na.
I have supplemented this sauce with essential amino acids (11130 Life Technologies),
vitamins (BME 100x, B6891 SIGMA), B27 diluted 1000 fold (Invitrogen), 1mM Sodium
Pyruvat, 1 mM Glutamine, 0,01 mM Glycine and water of course. To be sure that at
recording temperature (abouve 30°C) the pH will good you have to prepare a
HEPES/HEPES Na buffer separately, at higher concentration, to warm it up to 30-32
degrees and to adjust a pH with HEPES or HEPES Na.

In such solution hippocampal
dissociated cultures survived 5 days(I didn't try more) in thermostat at 35°C
without CO2, from 13DIV to 17DIV and I did not find that they differed from theirs
colleagues resided in MEM based medium in CO2 humidified incubator. Using this
extracellular medium I succeeded in recording of the dendrite motility, apparition
and disappearance of the spines, I saw the lateral diffusion of something
fluorescent coupled with NR2B subunits of the NMDA receptors. So it woks.