Primary Striatal Neuronal Culture - Drugs of Abuse: Neurological Reviews and Protocols - page 375

choline acetyltransferase-associated cholinergic cells and glial fi brillary acidic
protein-associated astrocytes (2) .

4. Notes

1. Although we use E19-E20 fetuses in most cases, postnatal d 1 rat pups have
also been used successfully with our cell preparation procedures (2) . However,
our experience is that the tissues from younger animals can provide more viable
cells and fewer non-neuronal cells. They seem to grow healthier and survive
longer as well.

2. Coating substrates, poly- D - or poly- L -lysine, can be purchased from Sigma in
sterile form over a range of molecular weights. The two isomers of polylysine
have a comparable cell adhesion promoting effect. However, L -isomer amino
acids seem to be biologically active; the D -isomer is usually used. We have had
good outcomes using poly- D -lysine (molecular weight = 70,000-150,000).

3. More careful control of using trypsin is important. Trypsin is a pH-sensitive
protease. It is almost completely inactive below pH 7. The length of time of using
this enzyme should be determined empirically and adjusted proportionately
according to the age of embryos. If substantial cell lysis occurs and the yield of
cells is not near expectations during dissociation, incubation duration of trypsin
should be shortened accordingly.

4. Caution is necessary when assessing cell viability by dye exclusion. Although
dye uptake marks cells that have grossly disrupted membranes, it may not detect
other forms of injury or an early stage of injury that affects cell adhesion or can
progress to cell death afterwards.

5. We use Ara-c as an antimitotic agent to reduce the population of non-neuronal
cells capable of DNA synthesis. To avoid possible toxic effects on neurons (6) ,
Ara-c is employed at its lowest effective concentration (5 M ). In addition, the

timing of addition of this agent is critical. It should be added into the medium
3-4 d after culturing when a confl uent monolayer of astroglial cells has formed,
which serves as a needed bed for neurons to grow a network of perikarya and
processes.

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6. Cultured cells seem to be very sensitive to air. Therefore, we usually exchange
three-fourths of the medium with a small amount of medium to be left to cover
the bottom of cell layer. This prevents cells from exposure to air and minimizes
the disturbance caused by feeding cultures with fresh medium. The optical
schedule for feeding cultures is once every 5-7 d in our case. This infrequent
feedings refl ect an unnecessary need to maintain high rates of cell proliferation,
and may benefi t cells with less disturbance that occurs between feedings.

7. Primary striatal cultures provide the closest approximation of the situation in
vivo, and it can be rationally employed for many purposes if one is aware of
some of its disadvantages as fresh tissues. They are heterogeneous. Thus, growth
of glial cells needs to be controlled by a mitotic inhibitor, and the phenotype
of neurons or fraction of a given phenotype of neurons needs to be identifi ed
immunohistochemically. Cell preparation procedures vary from experiment to