Caffeine is a popular psychostimulant drug that many people consume daily in the form of coffee, tea, sodas or energy drinks. It increases your alertness, reduces fatigue, and seems to enhance cognitive functions, but is also involved in triggering anxiety in certain individuals. Caffeine is a non-selective adenosine receptor antagonist. When stimulated the receptors facilitate neurotransmitter release, long term potentiation, smooth muscle relaxation, and vasodilation to mention a few. Immature cortical and hippocampal primary cell cultures from rat were treated day 1-4 in vitro with non-selective caffeine, the selective adenosine A2A antagonist istradefylline, and the selective adenosine receptor A2A agonist CGS21680. Preliminary results from counting the branching of the cortical cells seem to demonstrate that caffeine and istradefylline decrease the branching of neurite outgrowth in a dose-dependent manner. Caffeine and Istradefylline both also affected the overall neurite length in cortical cells. In addition, caffeine in its highest concentration affected the mitochondria function as well as the gene expression of MAO-B and tryptophan hydroxylase 2 in cortical cells. Furthermore, preliminary results also indicate that the highest concentration of caffeine also decreased in neurite length in hippocampal cells. To conclude, our results clearly demonstrate that caffeine may affect primary cell cultures as well as the neurotransmission in the brain. An impact on the brain that possibly may contribute to caffeins effect of anxiety disorders.