Species-specific vocalizations in mice have frequency-modulated (FM) components slower than the lower limit of FM direction selectivity in the core region of the mouse auditory cortex. To identify cortical areas selective to slow frequency modulation, we investigated tonal responses in the mouse auditory cortex using transcranial flavoprotein fluorescence imaging. For differentiating responses to frequency modulation from those to stimuli at constant frequencies, we focused on transient fluorescence changes after direction reversal of temporally repeated and superimposed FM sweeps. We found that the ultrasonic field (UF) in the belt cortical region selectively responded to the direction reversal. The dorsoposterior field (DP) also responded weakly to the reversal. Regarding the responses in UF, no apparent tonotopic map was found, and the right UF responses were significantly larger in amplitude than the left UF responses. The half-max latency in responses to FM sweeps was shorter in UF compared with that in the primary auditory cortex (A1) or anterior auditory field (AAF). Tracer injection experiments in the functionally identified UF and DP confirmed that these two areas receive afferent inputs from the dorsal part of the medial geniculate nucleus (MG). Calcium imaging of UF neurons stained with fura-2 were performed using a two-photon microscope, and the presence of UF neurons that were selective to both direction and direction reversal of slow frequency modulation was demonstrated. These results strongly suggest a role for UF, and possibly DP, as cortical areas specialized for processing slow frequency modulation in mice.

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Functional brain imaging using endogenous fluorescence of mitochondrial flavoprotein is useful for investigating mouse cortical activities via the intact skull, which is thin and sufficiently transparent in mice. We applied this method to investigate auditory cortical plasticity regulated by acoustic environments. Normal mice of the C57BL/6 strain, reared in various acoustic environments for at least 4 weeks after birth, were anaesthetized with urethane (1.7 g/kg, i.p.). Auditory cortical images of endogenous green fluorescence in blue light were recorded by a cooled CCD camera via the intact skull. Cortical responses elicited by tonal stimuli (5, 10 and 20 kHz) exhibited mirror-symmetrical tonotopic maps in the primary auditory cortex (AI) and anterior auditory field (AAF). Depression of auditory cortical responses regarding response duration was observed in sound-deprived mice compared with naïve mice reared in a normal acoustic environment. When mice were exposed to an environmental tonal stimulus at 10 kHz for more than 4 weeks after birth, the cortical responses were potentiated in a frequency-specific manner in respect to peak amplitude of the responses in AI, but not for the size of the responsive areas. Changes in AAF were less clear than those in AI. To determine the modified synapses by acoustic environments, neural responses in cortical slices were investigated with endogenous fluorescence imaging. The vertical thickness of responsive areas after supragranular electrical stimulation was significantly reduced in the slices obtained from sound-deprived mice. These results suggest that acoustic environments regulate the development of vertical intracortical circuits in the mouse auditory cortex.

The auditory thalamus and auditory cortex (AC) are pivotal structures in the central auditory system. However, the thalamocortical mechanisms of processing sounds are largely unknown. Investigation of this process benefits greatly from the use of mice because the mouse is a powerful animal model in which various experimental techniques, especially genetic tools, can be applied. However, the use of mice has been limited in auditory research, and thus even basic anatomical knowledge of the mouse central auditory system has not been sufficiently collected. Recently, optical imaging combined with morphological analyses has enabled the elucidation of detailed anatomical properties of the mouse auditory system. These techniques have uncovered fine AC maps with multiple frequency-organized regions, each of which receives point-to-point thalamocortical projections from different origins inside the lemniscal auditory thalamus, the ventral division of the medial geniculate body (MGv). This precise anatomy now provides a platform for physiological research. In this mini review article, we summarize these recent achievements that will facilitate physiological investigations in the mouse auditory system.

Endogenous fluorescence signals derived from mitochondria reflect activity-dependent changes in brain metabolism and may be exploited in functional brain imaging. Endogenous flavoprotein fluorescence imaging in mice is especially important because many genetically manipulated strains of mice are available and the transparent skull of mice allows transcranial fluorescence imaging of cortical activities. In the primary sensory areas of mice, cortical activities and experience-dependent plasticity have been investigated using transcranial fluorescence imaging. Furthermore, differential imaging, based on stimulus specificity of cortical areas, distinguished activities in higher visual areas around the primary visual cortex from those in primary visual cortex. The combination of transcranial fluorescence imaging with the suppression of cortical activities using photobleaching of flavoproteins is expected to aid in elucidating the roles of sensory cortices including higher areas in mice.