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Lineage Tracking and Differentiation of Neuro 2A cells (N2a) upon exposure to Dopamine, Monosodium Glutamate and Corticosterone

Cell to cell communication is critical for survival of an organism and it is achieved through signal transduction pathways. For the transmission of signal, molecules such as hormones can bind to a receptor on or inside the cell membrane. Dopamine (DA) a monoamine catecholamine neurotransmitter and hormone binds to dopamine receptor, inturn, affect movement, emotions and the reward system in the brain. The present study tested the hypothesis that Dopamine differentiates the N2a cells into neuronal lineage, and corticosterone (CORT) and monosodium glutamate (MSG) alter the differentiation in either way. Mouse neural crest‐derived cell line (Neuro2A cells, N2a cells) were treated with 10µM DA, MSG and CORT. Cells were seeded for different time points to track the lineage pattern of differentiation. Treatment was done for 24 hrs, 48 hrs, 72hrs and 96 hrs with respective control groups. Phase contrast microscopic images revealed that Dopamine treated cells showed neurite extensions and differentiation as time passed whereas CORT and MSG treatments suppressed the differentiation; the cells were round and showed signs of stress, cell death and autophagy. Immunocytochemical analysis with D1 receptor antibody indicated enhanced expression in the DA groups and diminished D1 receptor expression in CORT and MSG groups. Furthermore, quantitative analysis revealed that D1 receptor expression was significantly enhanced in DA group compared to the control group (medium containing ascorbic acid and phosphate buffered saline) and untreated control group (medium only). Interestingly D1 receptor expression in the DA group was significantly upregulated compared to the CORT group but not significantly different in the MSG group. Furthermore, inorder to assess apoptosis AnnexinV/Propidium Iodide (PI) staining was done using Fluorescence Assorted single cell sorter (FACS) Analysis. Immunolabeling with Caspase‐3 marker gene was done to detect apoptosis. N2a cells were treated with DA, CORT and MSG at 10 µM concentration for 24 hrs. Propidium iodide labelled cells that were already dead or in the late stage of apoptosis whereas Annexin V bound to the cells early in apoptosis and continued to be bound through cell death. Counting of the cells showed that MSG induced 1.8 % apoptosis, CORT induced 1.2 % and DA induced 1.1%, as against control group cells of 0.7 %. The total cell death including apoptosis and necrosis was highest in the MSG group (3.4%) compared to the control (1.7 %), DA (2.1%) and CORT (1.9%) treated groups. This data suggest differential effects elicited by DA (happy hormone) versus stress hormone. Our results also raise the question whether DA induces catecholaminergic pathway of differentiation, CORT stimulates glial type differentiation and MSG stimulates glutamate or GABA differentiation ‐ questions to be answered in future studies.