More dendritic spine synapses occur on mature neurons in hippocampal slices by 2 h of incubation in vitro, than in perfusion-fixed hippocampus. What conditions initiate this spinogenesis and how rapidly do the spines begin to proliferate on mature neurons? To address these questions, CA1 field of the hippocampus neurons expressing green fluorescent protein in living slices from mature mice were imaged with two-photon microscopy. Spines disappeared and dendrites were varicose immediately after slice preparation in ice-cold artificial cerebrospinal fluid (ACSF). Electron microscopy (EM) revealed disrupted dendritic cytoplasm, enlarged or free-floating postsynaptic densities, and excessive axonal endocytosis. Upon warming dendritic varicosities shrank and spines rapidly reappeared within a few minutes illustrating the remarkable resilience of mature hippocampal neurons in slices. When membrane impermeant sucrose was substituted for NaCl in ACSF dendrites remained spiny at ice-cold temperatures and EM revealed less disruption. Nevertheless, spine number and length increased within 30 min in warm ACSF even when the extracellular calcium concentration was zero and synaptic transmission was blocked. When slices were first recovered for several hours and then chilled in 6 degrees C ACSF many spines disappeared and the dendrites became varicose. Upon re-warming varicosities shrank and spines reemerged in the same position from which they disappeared. In addition, new spines formed and spines were longer suggesting that chilling, not the initial injury from slicing, caused the spines to disappear while re-warming triggered the spine proliferation on mature neurons. The new spines might be a substrate for neuronal recovery of function, when neurons have been chilled or exposed to other traumatic conditions that disrupt ionic homeostasis.