We used autofluorescence of mitochondrial flavoproteins to image cortical neural activity in the rat. Green autofluorescence in blue light was examined in slices obtained from rat cerebral cortex. About half of the basal autofluorescence was modulated by the presence or absence of O2 or glucose in the medium. Repetitive electrical stimulation at 20 Hz for 1 s produced a localized fluorescence increase in the slices. The amplitude of the increase was 27 +/- 2 % (mean +/- S.D., n = 35). Tetrodotoxin or diphenyleneiodonium, an inhibitor of flavoproteins, blocked the autofluorescence responses. The autofluorescence responses were not observed in slices perfused with calcium-, glucose- or O2-free medium. In the primary somatosensory cortex of rats anaesthetized with urethane (1.5 g kg-1, I.P.), an activity-dependent increase in autofluorescence of 20 +/- 4 % (n = 6) was observed after electrical cortical stimulation at 100 Hz for 1 s, and an increase of 2.6 +/- 0.5 % (n = 33) after vibratory skin stimulation at 50 Hz for 1 s applied to the plantar hindpaw. These responses were large enough to allow visualization of the neural activity without having to average a number of trials. The distribution of the fluorescence responses after electrical or vibratory skin stimulation was comparable to that of the cortical field potentials in the same rats. The fluorescence responses were followed by an increase in arterial blood flow. The former were resistant to an inhibitor of nitric oxide synthase, while the latter was inhibited. Thus, activity-dependent changes in the autofluorescence of flavoproteins are useful for functional brain imaging in vivo.

The preservation of visual property differences among the higher visual areas following V1 lesions and their loss following SC lesions indicate that pathways from the SC through the thalamus to higher cortical areas are sufficient to support these differences.

The primary auditory cortex (AI) is the representative recipient of information from the ears in the mammalian cortex. However, the delineation of the AI is still controversial in a mouse. Recently, it was reported, using optical imaging, that two distinct areas of the AI, located ventrally and dorsally, are activated by high-frequency tones, whereas only one area is activated by low-frequency tones. Here, we show that the dorsal high-frequency area is an independent region that is separated from the rest of the AI. We could visualize the two distinct high-frequency areas using flavoprotein fluorescence imaging, as reported previously. SMI-32 immunolabeling revealed that the dorsal region had a different cytoarchitectural pattern from the rest of the AI. Specifically, the ratio of SMI-32-positive pyramidal neurons to nonpyramidal neurons was larger in the dorsal high-frequency area than the rest of the AI. We named this new region the dorsomedial field (DM). Retrograde tracing showed that neurons projecting to the DM were localized in the rostral part of the ventral division of the medial geniculate body with a distinct frequency organization, where few neurons projected to the AI. Furthermore, the responses of the DM to ultrasonic courtship songs presented by males were significantly greater in females than in males; in contrast, there was no sex difference in response to artificial pure tones. Our findings offer a basic outline on the processing of ultrasonic vocal information on the basis of the precisely subdivided, multiple frequency-organized auditory cortex map in mice.

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.

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.

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Optical imaging studies have recently revealed the presence of multiple auditory cortical regions in the mouse brain. We have previously demonstrated, using flavoprotein fluorescence imaging, at least six regions in the mouse auditory cortex, including the anterior auditory field (AAF), primary auditory cortex (AI), the secondary auditory field (AII), dorsoanterior field (DA), dorsomedial field (DM), and dorsoposterior field (DP). While multiple regions in the visual cortex and somatosensory cortex have been annotated and consolidated in recent brain atlases, the multiple auditory cortical regions have not yet been presented from a coronal view. In the current study, we obtained regional coordinates of the six auditory cortical regions of the C57BL/6 mouse brain and illustrated these regions on template coronal brain slices. These results should reinforce the existing mouse brain atlases and support future studies in the auditory cortex.

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