<p>Neonicotionoid pesticides are insecticides which have been thought to be one of the causes of Colony Collapse Disorder. They were considered to be less toxic to mammals than insects, but some of the recent studies suggested their association with disruption of higher brain fuction in mammals. Nevertheless, it is still unclear how neonicotionoids affect on the central nervous system. Here, we propose the use of in vivo Ca²⁺ imaging with two-photon microscope to detect abnormal activity of neuronal circuits in brain with the application of neonicotionoids.</p><p>In this study, a less than the no-observed-adverse-effect level (NOAEL) of acetamiprid (20 mg/kg bw); and a tenth or half of the median lethal doses of nicotine (0.33 or 1.65 mg/kg bw respectively) were orally administered to mice. They were subjected to elevated plus maze test and Ca²⁺ imaging by two-photon microscope in the somatosensory cortex. We further detected acetamiprid and metabolites in brain and blood an hour after the administration.</p><p>Mice exposed to acetamiprid or nicotine exhibited an increase in anxiety-like behavior that associated with the altered activities of the neuronal population in the somatosensory cortex. Although the dose of acetamiprid used in the current study was below the NOAEL, both acetamiprid and nicotine affected the behavior and the neuronal activity in the somatosensory cortex. The results suggested that in vivo Ca²⁺ imaging using two-photon microscope enabled highly sensitive detection of brain neurodisruption by neurotoxicants.</p>