Journal Article Imaging of neuronal activity in awake mice by measurements of flavoprotein autofluorescence corrected for cerebral blood flow

Manami, Takahashi  ,  Urushihata, Takuya  ,  Takuwa, Hiroyuki  ,  Sakata, Kazumi  ,  Takado, Yuhei  ,  Shimizu, Eiji  ,  Suhara, Tetsuya  ,  Higuchi, Makoto  ,  Ito, Hiroshi

11 ( 723 ) 2018-01
Green fluorescence imaging (e.g., flavoprotein autofluorescence imaging, FAI) can beused to measure neuronal activity and oxygen metabolism in living brains withoutexpressing fluorescence proteins. It is useful for understanding the mechanism ofvarious brain functions and their abnormalities in age-related brain diseases. However,hemoglobin in cerebral blood vessels absorbs green fluorescence, hampering accurateassessments of brain function in animal models with cerebral blood vessel dysfunctionsand subsequent cerebral blood flow (CBF) alterations. In the present study, we developeda new method to correct FAI signals for hemoglobin-dependent green fluorescencereductions by simultaneous measurements of green fluorescence and intrinsic opticalsignals. Intrinsic optical imaging enabled evaluations of light absorption and scattersby hemoglobin, which could then be applied to corrections of green fluorescenceintensities. Using thismethod, enhanced flavoprotein autofluorescence by sensory stimuliwas successfully detected in the brains of awake mice, despite increases of CBF,and hemoglobin interference. Moreover, flavoprotein autofluorescence could be properlyquantified in a resting state and during sensory stimulation by a CO2 inhalation challenge,which modified vascular responses without overtly affecting neuronal activities. Theflavoprotein autofluorescence signal data obtained here were in good agreement with theprevious findings from a condition with drug-induced blockade of cerebral vasodilation,justifying the current assaying methodology. Application of this technology to studies onanimal models of brain diseases with possible changes of CBF, including age-relatedneurological disorders, would provide better understanding of the mechanisms ofneurovascular coupling in pathological circumstances.

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