Neurons exhibit stimulation-induced ultrastructural changes such as increase of thickness and curvature of the postsynaptic density, decrease in contact area between subsurface cistern and plasma membrane, and formation of CaMKII clusters and synaptic spinules. These structural characteristics help in identifying the activity state of the neuron and should be taken into consideration when interpreting ultrastructural features of the neurons. Here in organotypic hippocampal slice cultures where experimental conditions can be easily manipulated, two additional features are documented in forebrain neurons as reliable benchmarks for stimulation-induced structural changes: (1) The neuronal nucleus showed conspicuous clustering of dark chromatin, and (2) the endoplasmic reticulum formed stacks with a uniform gap of ~ 13 nm filled with dark materials. Both structural changes progressed with time and were reversible upon returning the slice cultures to control medium. These stimulation-induced structural changes were also verified in dissociated hippocampal neuronal cultures and perfusion-fixed brains. In hippocampal slice cultures, the neuronal chromatin clustering was detectable within 30 s of depolarization with high K+ (90 mM) or treatment with NMDA (50 μM). In contrast, the formation of ER cisternal stacks did not become apparent for another 30 s. Importantly, in dissociated neuronal cultures, when the extracellular calcium was chelated by EGTA, treatment with high K+ no longer induced these changes. These results indicate that the stimulation-induced chromatin clustering and formation of ER stacks in neurons are calcium-dependent. Additionally, mitochondria in neuronal somas of tissue culture samples consistently became swollen upon stimulation. However, swollen mitochondria were also present in some neurons of control samples, but could be eliminated by blocking basal activity or calcium influx. This calcium-dependent structural change of mitochondria is specific to neurons. These structural changes may bring insights to the neuron's response to intracellular calcium rise upon stimulation.