Tonotopic mapping in the in vivo mouse via high resolution fMRI
Guilherme Blazquez Freches1, Cristina Chavarrias1, and Noam Shemesh1

1Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Lisbon, Portugal


The rodent auditory system has been a popular research subject for electrophysiological studies for its complexity, fine tuning and adaptability. More recently, some studies on auditory Functional Magnetic Resonance Imaging (fMRI) in rats have surfaced, aiming to unravel this system’s intricacies by capturing whole brain activity noninvasively. Auditory mapping in the mouse could be highly valuable given its importance vis-à-vis transgenic models and optogenetics. This study provides the first tonotopic mapping in the in vivo mouse via high resolution fMRI. We demonstrate robust activation in the auditory pathway, and specific tonotopy in several prominent regions along the pathway.


Whereas human tonotopy has been extensively studied via fMRI [1-4], rodent tonotopy is still in its infancy. Rat experiments have been recently presented [5-12], but –to our knowledge– auditory mapping in mice has never been performed. We here studied the mouse auditory pathway in-vivo, and in particular, BOLD-derived tonotopic maps.


Sound delivery system: A dedicated system for delivering accurate auditory stimuli was custom designed. Briefly, the system consisted of: (1) a Yamaha AG-03 soundboard allowing playback of sounds up to 96 kHz (sample rate of 192 kHz) with a dynamic range of 24 bits and 2.451 V RMS output before clipping; (2) an in-house designed voltage amplifier capable of a 3x amplification up to 24 Vpp; (3) L010 Kemo piezoelectric speaker capable of producing ultrasonic sounds at high output levels (above 100 dB) with a relatively flat frequency response up to 75 kHz. The emitted sounds were then delivered via custom tubing involving nylon and tygon tubes, interfaced with custom 3D-printed PET parts to a single ear. A Brüel and Kjær 4939-A-011, free-field microphone with a Type 2670 preamplifier was connected directly into the Yamaha Soundboard and placed 1 mm away from the auditory setup to calibrate and test the system’s performance and to record scanner-induced sounds. MRI setup: All experiments were pre-approved by the Institute’s animal ethics committee. Experiments were performed on a Bruker BioSpec 9.4T scanner with a 20 cm bore and gradients capable of producing up to 66 G/cm in all directions. For transmittance, an 86 mm volume resonator was used, whereas a 4-channel array cryocoil was used for signal detection. Male mice (c57bl/6, N=6) weighing approximately 25 gr were briefly anesthetized with 4% isoflurane and then quickly placed under a medetomidine sedation regime (bolus 0.4 mg/kg then 0.8 mg/kg/h, continuous infusion) as described in [13], whereupon the isoflurane was discontinued. Animal physiology including transcutaneous CO2, rectal temperature, and breathing rate were constantly monitored. The animals breathed air enriched with Oxygen (27%) measured by a oxygen Sensor (Viamed, United Kingdom) throughout the experiments. One ear was carefully sealed with wax and the Tygon tube was placed within the other ear, allowing the delivery of auditory stimuli preferentially to that ear. fMRI experiments and analysis: Following localization scans, fMRI experiments were conducted using an ultrafast FISP sequence with the following parameters: TR/TE = 2.8/1.4 ms, slice thickness = 650 μm, FOV = 15x15 mm2, matrix size 100x100 leading to an in-plane resolution of 150x150 (μm)2, and a 1.307 seconds scan time per volume. The fMRI paradigm consisted of 45 seconds of rest, followed by five blocks of {15 seconds of auditory stimulation, 45 seconds of rest}, for a total time of 7.5 min per scan. The auditory stimulus consisted of a pure tone of 5, 12 or 20kHz; animals were allowed to rest between paradigms for ~3 minutes. The data were analyzed using SPM® (called through fMRat [14]) for mapping activation in the mouse brain).

Results and Discussion

Figure 1 shows the spectral analysis of the FISP sounds as well as the frequencies delivered to the ear of the mouse. The 5kHz stimulation show the most (unwanted) harmonics. The FISP noise, while significant, was still at least 20dB lower compared to the stimuli given, and hence was considered not to significantly affect the experiment. Figure 2 shows raw data from the experiment, including a volume from the FISP experiments (upper panel) and signal time course from a representative experiment (one single run in a single rat) for three selected ROIs within the auditory pathway (lower panel). Excellent SNR was documented, and the BOLD response can be observed with the naked eye. Figure 3 shows t-maps for each frequency; notably, nearly the entire auditory pathway is highlighted, including its major relays: Auditory nerve, Superior Olivary Complex (SOC), Lateral Lemniscus (LL), Inferior Colliculus (IC), and Medial Geniculate Nucleus (MGB), with t-statistics as high as t=19.3. Auditory Cortex (AC) was not demonstrated in these maps, although ROI analysis (not shown) suggests it may be active, but with different BOLD dynamics, thereby reducing its significance levels in Figure 3. Figure 4 shows tonotopic maps in the more posterior slices, where activation was most prominent. Tonotopy was observed in several regions, most notably in IC, where three layers show a clear tonotopic pattern going from dorsolateral regions to medioventral ones with increasing frequency, confirming the electrophysiological findings in mice [15] and fMRI in rats [7]. This study thus bodes well for auditory pathway mapping in the in-vivo mouse in models of plasticity / aberrations.


The authors acknowledge funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Starting Grant, agreement No. 679058). We thank Dr. Daniel Nunes for technical assistance.


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Frequency Spectra of the sounds present in this experiment (Left). The FISP frequency spectrum peaks at about 90 dB between 2 and 9 kHz and all the fundamentals of the sounds deployed to the mouse were above this plateau. Unfortunately, some of the harmonics (especially in the 5 and 12 kHz) were still high (e.g the first harmonic of the 5kHz tone peaks at 90 dB) but always at least 6 dB lower than the fundamental. Closeup of the frequency spectra from 0 to 20 kHz (Right).

Example of the functional volumes acquired (150x150 μm2 in-plane resolution) without any preprocessing (Upper) Example of the raw signal (only detrended) for the Auditory Nerve, Ventral IC and Lateral Lemniscus with stimulations marked in red for one run in one mouse. The high SNR of the raw signal is evidenced

Activation maps for the 3 frequencies employed in this study. Tonotopy along the auditory pathway, as well as, more specifically in the IC can be observed. Activation can be seen in all structures of the auditory pathway (especially for lower frequencies) , AN, SOC,LL, IC and MGB with very high t-values. The plotted activations represent a p-value of p<0.001.

Tonotopy reflecting BOLD variations in all mice studied. This map shows a very distinct tonotopic pattern with high frequency specific voxels being located ventromedialy and lower frequency voxels being located more dorsolaterally. This is excelent agreement with the existing literature for rodents, both with electrophysiological studies[15] and with fMRI[7]. Additionaly, tonotopy can be seen in the SOC and LL, in agreement with electrophysiological studies [15]. Such maps can be used in the future to map functional plasticity in-vivo.

Proc. Intl. Soc. Mag. Reson. Med. 25 (2017)