Enriched Neuronal Cultures Enable More Accurate Assessment of Receptor Expression and Enhance Growth of Neuronal Processes

Lizeth Martinez, See Yang, and Kiowa BowerDepartment of Natural Sciences and Mathematics, Dominican University of California, San

Rafael, CA 94901AbstractWe are studying the changes in receptor expression levels as embryonic stem cells  (ESCs) differentiate into neurons by adding AraC ,a selective inhibitor of DNA synthesis, and inducing them in a monolayer fashion.  We can identify which cells in our culture are neurons by using MAP2 as a neuronal marker . Our results indicate that this protocol yields higher percentages of neurons and thus more reliable results from our gene-expression studies.  Because reduced glial cell proliferation leaves more space for the growth of the developing neurons, we examined the potential effect of the new conditions on neuron morphology.  Using immunocytochemistry to visualize extent of axon and dendrite growth, we found that the new conditions led to increased numbers of these neuronal processes.

Background• Neuroreceptors are membrane

proteins that are activated by chemical signals from other cells, and are most abundant at the synapses between neurons.  

• These receptors are critical for neuron communication and some play a role in neuronal differentiation during the embryonic development of the nervous system.  

• P19 embroynal carcinoma cells are an effective model system for investigating neuronal differentiation because their development, morphology, and biochemistry are similar to in vivo differentiation of neurons.  Materials and Methods

• The ESCs are grown in a growth media with 5% fetal bovine serum

• The cells become induced by retionic acid in a monolayer method using a T75 confluent flask. Induction occurs for 4 days.

• Induced cells are seeded on 12 well plates with glass slides, coated with proteins.

• AraC is added after 3 days of seeding.

Results• Visible changes in cell morphology

after 1 day• Cells appear dead & lysed but some

cell components remain attached

• Immunocytochemistry

DNA (Nucleus) MAP 2

Synapsin Merged

Day of drug addition 1 day after addition of drug

Conclusions• Mitotic inhibitors are effective

Validity of receptor expression studies• Monolayer induction better

than EB protocolEasier methodologyConsistency of neuron populationsMore numerous dendrites• Potentially useful for

synaptogenesis studies• Identified expression

changes in several neuroreceptors