Background. The integrity of neuronal axons and ability to differentiate are of utmost importance to understand pathology of neurological diseases. β-actin is one of the cytoskeletal proteins that is important for cell integrity and synaptic formation. Previous studies showed contradictory findings concerning the effects of β-amyloid protein on neuronal structure and synaptic function. N-methyl-D-aspartate receptors (NMDARs) have crucial role in neuronal plasticity. Neuronal cell line (H19-7) generated from rat embryonic hippocampal neurons offers a model for screening assays. Objective. In this study, we used microculture system to characterize H19/7 cells in regard to their phenotypes, differentiation, synaptic connections, responsiveness to temperature and insulin like growth factor-I (IGF-I) hormone, and the ability to express growth hormone receptors (GHRs). Moreover, we examined the ability of the differentiated H19/7 cells to coexpress β amyloid and β actin with partners of NMDARs. Results. We found that raising temperature of cell culture to 39°C in medium supplemented with high glucose, basic fibroblast growth factors (bFGF) and IGF-I decreased the rapid cell proliferation and induced differentiation seen as elongation, multiple processes extension, thickening of cell bodies, and formation of synaptic connections. All differentiated cells express GHRs highlighting the importance of growth factors in maintaining the integrity of H19/7 cells. We further proved the differentiation of cells by testing the expression of NSE using western blots at different temperature and days of development. We found significantly higher expression seen at days 4 and 8 at 39°C compared to day 4 at 34°C. We found that H19/7 cells express cytoskeletal protein; β-actin as well as neuronal cytoplasmic proteins including, β-amyloid, and neuron specific enolase (NSE); two key molecular marker of neuronal cells. Moreover, we showed that differentiated H19/7 cells express a channel protein NR1and NR2A; partners of N-methyl-D-aspartate receptors. We found that immune-reactivity of β-actin, and NSE was uniformly distributed throughout the cytoplasm in soma and neuronal processes. All differentiated cells showed co-expression of both β actin and NR2A; β amyloid and NR1 proteins especially on soma and neuronal processes suggesting a role of β actin and β amyloid in NMDAR-mediated effects at synaptic connection sites. Conclusion. This study suggested that H19/7 cells represent promising cell line model that reveal mechanisms regulating neuronal axon outgrowth and integrity that may help in treatment of neurological diseases in the future.