In this chapter, flavoprotein fluorescence imaging is described, with a focus on transcranial imaging for investigating experience-dependent plasticity in mice. The molecular mechanisms underlying higher brain functions are important subjects of neuroscience research. The cerebral cortex, which is expected to have essential roles in higher functions, has mainly been investigated in primates. However, the analytical methods for elucidating molecular mechanisms in the primate brain are rather limited. An alternative approach is to investigate higher cortical functions in rodents. In particular, mice are very useful for investigating molecular mechanisms, because many genetically manipulated strains are available. However, the cortical functions of rodents can not be studied intensively, partly because the cortex is fragile, and electrophysiological analysis is sometimes difficult. In contrast, mice have thin skulls that are transparent enough to allow transcranial imaging of the cortical activities through the intact skull (Schuett et al. 2002, Shibuki et al. 2007). Auditory, visual, and somatosensory cortices are visible through the intact skull (Figure 7.1A). Transcranial analysis of cortical functions has a number of technical merits: the operation to remove the skull is omitted so that surgical damages on the cortex can be avoided, and the fragile cortex of mice is kept intact within the skull during the recording experiments. Optical imaging of cortical activities usually requires experimental skills, whereas the transcranial imaging of the mouse cortical activities may be performed even by beginner neuroscientists or students.
In transcranial analysis of cortical functions, application of exogenous indicators, such as voltage-sensitive dyes or calcium indicators, is difficult. Transcranial imaging may be performed in mice that express with protein sensors derived from GFP (McGann et al. 2005, Diez-Garcia et al. 2007). Alternatively, it may be performed using some intrinsic signals reflecting cortical activities. Most of intrinsic signals reflecting brain activities are coupled with activity-dependent facilitation of aerobic energy metabolism (Fein and Tsacopoulos 1988, Shibuki 1989, 1990, Vanzetta and Grinvald 1999). One of the results of facilitated energy metabolism is that oxyhemoglobin in the capillary is converted to deoxyhemoglobin, and that the light absorption properties of hemoglobin are changed (Frostig et al. 1990). Endogenous fluorescence changes are also produced by facilitated energy metabolism in the brain (Chance et al. 1962), because fluorescence substances such as NADH or flavoproteins are involved in aerobic energy metabolism (Figure 7.1B).
Activity-dependent changes in endogenous fluorescence derived from NADH have been used to monitor brain activities (Chance et al. 1962, Rosenthal and Jöbsis 1971, Lothman et al. 1975, Lewis and Schuette 1976). However, decreases in fluorescence signals derived from NADH might also be caused by activity-dependent increases in blood flow (Kitaura et al. 2007), because both excitation light and emitted fluorescence are easily absorbed by hemoglobin. Although activity-dependent increases in flavoprotein fluorescence signal are unlikely to be mimicked by hemodynamic responses, flavoprotein fluorescence changes had not been used to monitor brain activities in previous studies, as these signals were not clearly recorded (Aubin et al. 1979). Endogenous fluorescence derived from NADH or flavoprotein is weak compared with that derived from fluorescence dyes. Therefore, clear separation of the faint fluorescence from the strong excitation light is essential. The faint fluorescence must be detected by a sensitive camera with a low noise level. Therefore, application of endogenous fluorescence derived from flavoproteins to functional brain imaging requires recent development of advanced optical devices (Shibuki et al. 2003, Reinert et al. 2004, Coutinho et al. 2004, Weber et al. 2004, Husson et al. 2007).
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