Auditory cortical areas activated by slow frequency-modulated sounds 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

Subdivisions of MG projecting to the auditory cortex.

<p>(<b>A</b>) Cresyl violet staining of MG. (<b>B</b>) SMI-32 immunostaining of MG in the adjacent section. (<b>C</b>) BDA staining of MG after BDA injection into the cortical area responding to a 25 kHz AM sound within A1. (<b>D</b>) BDA staining of MG after BDA injection into the cortical area responding to a 25 kHz AM sound within AAF. (<b>E</b>) BDA staining of MG after BDA injection into UF. (<b>F</b>) BDA staining of MG after BDA injection into DP. Sections were approximately 3.0 mm posterior to bregma.</p

Absence of tonotopic maps in UF.

<p>(<b>A</b>) Cortical responses to FM direction reversal from upward to downward recorded in a mouse. The frequency ranged 5–11 (left), 15–21 (middle), or 25–31 kHz (right). The same responding area that approximately corresponded to UF is marked with dotted lines to assist comparison of responses shown in each panel. (<b>B</b>) The spatial distribution of response peaks to FM direction reversal from upward to downward in UF of 6 mice (circles). The relative locations of the response peaks to a 20 kHz AM sound in A1 (asterisk) are shown. (<b>C</b>) The spatial distribution of response peaks to FM direction reversal from downward to upward in UF of the same 6 mice. (<b>D</b>) Response amplitudes to FM direction reversal from upward to downward, and downward to upward for each frequency range.</p

Responses to FM direction reversal.

<p>(<b>A</b>) Responses to FM direction reversal from upward to downward. Inset shows the stimulus condition used for producing the responses. Time after the direction reversal is shown in each panel. The same areas that approximately corresponded to UF and DP are marked with dotted lines to assist comparison of responses shown in (<b>A</b>–<b>C</b>). The schematic drawing shows superimpused FM sweeps. (<b>B</b>) Responses to direction reversal from downward to upward. (<b>C</b>) Responses to a 20 kHz AM sound. Responses in (<b>A–C</b>) were recorded from the same mouse. (<b>D</b>) Time courses for fluorescence changes in response to FM direction reversal from upward to downward recorded in A1, AAF, UF and DP. Mean and S.E.M obtained from 13 mice are shown. The image shows the windows in A1, AAF, UF and DP at which fluorescence changes were measured. The peak amplitude in UF was significantly larger than those in DP, A1 and AAF (P<0.002, respectively). (<b>E</b>) Time courses for fluorescence changes in response to FM direction reversal from downward to upward. The peak amplitude in UF was significantly larger than those at DP, A1 and AAF (P<0.002, respectively). (<b>F</b>) Cortical responses to direction reversal of randomly-spaced FM sweeps from upward to downward. (<b>G</b>) Responses to direction reversal of randomly-spaced FM sweeps from downward to upward.</p

FM sweep speeds and cortical responses in UF and DP.

<p>(<b>A</b>) Response amplitudes to direction reversal of FM sweeps at a speed between 2 and 120 kHz/s from upward to downward in UF and DP of 6 mice. Insets show image examples. At a speed of 2 or 120 kHz/s, response amplitudes in UF and DP were significantly smaller than the corresponding values obtained at 24 kHz/s (P<0.004, respectively). (<b>B</b>) Response amplitudes to FM direction reversal between 2 and 120 kHz/s from downward to upward in UF and DP of the same 6 mice. At a speed of 2 or 120 kHz/s, response amplitudes in UF and DP were significantly smaller than the corresponding values obtained at 24 kHz/s (P<0.004, respectively).</p

Comparison of responses to FM direction reversal between hemispheres.

<p>(<b>A</b>) Cortical responses to FM direction reversal from upward to downward in the right (left panel) and left (right panel) hemispheres. The responding areas that approximately corresponded to UF and DP are marked with dotted lines to show the symmetrical relation between both hemispheres. (<b>B</b>) Response amplitudes to FM direction reversal in UF and DP of both hemispheres. Response amplitudes to a 20 kHz AM sound in A1 and AAF of both hemispheres are also shown. Mean and S.E.M were obtained for 13 hemispheres for each. Significant differences between hemispheres were found only regarding the UF responses to FM direction reversal from upward to downward (P<0.05), and downward to upward (P<0.02).</p

Responses to tonal stimuli in the auditory cortex.

<p>(<b>A</b>) Responses to AM sounds at 5 kHz (upper), 10 kHz (middle) and 20 kHz (lower panels). Time after stimulus onset is shown in each panel. Inset shows schematic drawing of the fluorescence responses to AM sounds mainly in A1, AAF and A2. (<b>B</b>) Responses to upward (upper panels) and downward (lower panels) FM sweeps ranging between 5 and 40 kHz for 0.5 s. Inset shows the fluorescence responses to FM sweeps in a large area including A1, A2, AAF, UF and DP. Images in (<b>A</b>) and (<b>B</b>) were obtained from the same mouse. The schematic outlines of A1, AAF and A2 are superimposed on the images.</p

Half-max latency for cortical responses to FM sweeps.

<p>(<b>A</b>) Window (0.26 by 0.26 mm) position for measuring half-max latency to FM sounds ranging from 5 kHz to 40 kHz, and from 40 kHz to 5 kHz for a period of 0.5 s. Windows are shown on the image with the response to a 20 kHz AM sound. Black spots show the response peaks in A1 and AAF. W11 was located at the crossing point of an anterior-posterior line passing through the peak in AAF and a medial-lateral line passing through the peak in A1. The cortical area around UF was covered by the 36 windows (W11∼W66). (<b>B</b>) Half-max latency measured at W11, W13, W41 and W43. The latency at W43 was significantly shorter than that at W11, W13 or W41 (P<0.03, respectively). Responses to FM sounds ranging from 5 kHz to 40 kHz, and from 40 kHz to 5 kHz were averaged to reduce variability in data. (<b>C</b>) Time courses of fluorescence responses at W41∼W46. Inset shows twice-expanded traces around the half amplitudes. (<b>D</b>) Time courses of fluorescence responses at W13∼W63.</p

Calcium imaging of UF neurons.

<p>(<b>A</b>) Two photon image of cell bodies in UF. Calcium signals were obtained from green neuronal cell bodies stained with fura-2 but not SR101. (<b>B</b>) Neuronal responses selective to FM direction reversal from upward to downward (left), or downward to upward (middle). Right traces were recorded in a neuron responding to FM direction reversal with no apparent preference. Decreases in ΔF/F₀ (or calcium increases) were plotted for the upward direction. (<b>C</b>) Relationship between the response amplitudes to FM direction reversal from upward to downward, and from downward to upward in 417 neurons. (<b>D</b>) Selectivity index based on the response amplitudes shown in (<b>C</b>). (<b>E</b>) Relationship between the selectivity index for FM direction reversal and that for FM direction in 87 neurons.</p