In vivo transcranial flavoprotein autofluorescence imaging of tonotopic map reorganization in the mouse auditory cortex with impaired auditory periphery.

Ototoxic-drug-induced hearing disturbances in the auditory periphery are associated with tonotopic map reorganization and neural activity modulation, as well as changes in neural correlates in the central auditory pathway, including the auditory cortex (AC). Previous studies have reported that peripheral auditory impairment induces AC plasticity that involves changes in the balance of excitatory vs. inhibitory synapses, within existing and newly forming patterns of connectivity. Although we know that such plastic changes modulate sound-evoked neural responses and the organization of tonotopic maps in the primary AC (A1), little is known about the effects of peripheral impairment on other frequency-organized AC subfields, such as the anterior auditory field (AAF) and the secondary auditory cortex (A2). Therefore, to examine ototoxic-drug-induced spatiotemporal effects on AC subfields, we measured sound-evoked neural activity in mice before and after the administration of kanamycin sulfate (1 mg/g body weight) and bumetanide (0.05 mg/g body weight), using in vivo transcranial flavoprotein autofluorescence imaging over a 4-week period. At first, ototoxic treatment gradually reduced responses driven by tone bursts with lower- (≤8 kHz) and middle- (e.g., 16 kHz) range frequencies in all AC subfields. Subsequently, response intensities in the A1 recovered to more than 78% of the pre-drug condition; however, in the AAF and A2, they remained significantly lower and were unchanged over 3 weeks. Furthermore, after drug administration, the best frequency (BF) areas of the lower (4 and 8 kHz) and higher (25 and 32 kHz) ranges in all subfields were reduced and shifted to those of a middle range (centered around 16 kHz) during the 3 weeks following drug administration. Our results also indicated that, compared with A1, BF distributions in the AAF and A2 were sharper around 16 kHz 3 weeks after drug administration. These results indicate that the ototoxic-damage-induced tonotopic map reorganizations that occurred in each of the three AC subfields were similar, but that there were subfield-dependent differences in the extent of response intensities and in the activated areas that were responsive to tone bursts with specific frequencies. Thus, by examining cortical reorganization induced by ototoxic drugs, we may contribute to the understanding of how this reorganization can be caused by peripheral damage.

Flavoprotein fluorescence imaging-based electrode implantation for subfield-targeted chronic recording in the mouse auditory cortex.

BACKGROUND: Chronic neural recording in freely moving animals is important for understanding neural activities of cortical neurons associated with various behavioral contexts. In small animals such as mice, it has been difficult to implant recording electrodes into exact locations according to stereotactic coordinates, skull geometry, or the shape of blood vessels. The main reason for this difficulty is large individual differences in the exact location of the targeted brain area. NEW METHODS: We propose a new electrode implantation procedure that is combined with transcranial flavoprotein fluorescence imaging. We demonstrate the effectiveness of this method in the auditory cortex (AC) of mice. RESULTS: Prior to electrode implantation, we executed transcranial flavoprotein fluorescence imaging in anesthetized mice and identified the exact location of AC subfields through the skull in each animal. Next, we surgically implanted a microdrive with a tungsten electrode into exactly the identified location. Finally, we recorded neural activity in freely moving conditions and evaluated the success rate of recording auditory responses. COMPARISON WITH EXISTING METHOD(S): These procedures dramatically improved the success rate of recording auditory responses from 21.1% without imaging to 100.0% with imaging. We also identified large individual differences in positional relationships between sound-driven response areas and the squamosal suture or blood vessels. CONCLUSIONS: Combining chronic electrophysiology with transcranial flavoprotein fluorescence imaging before implantation enables the realization of reliable subfield-targeted neural recording from freely moving small animals.

Micro-coil-induced Inhomogeneous Electric Field Produces sound-driven-like Neural Responses in Microcircuits of the Mouse Auditory Cortex In Vivo.

Magnetic stimulation is widely used in neuroscience research and clinical treatment. Despite recent progress in understanding the neural modulation mechanism of conventional magnetic stimulation methods, the physiological mechanism at the cortical microcircuit level is not well understood due to the poor stimulation focality and large electric artifact in the recording. To overcome these issues, we used a sub-millimeter-sized coil (micro-coil) to stimulate the mouse auditory cortex in vivo. To determine the mechanism, we conducted the first direct electrophysiological recording of micro-coil-driven neural responses at multiple sites on the horizontal surface and laminar areas of the auditory cortex. The laminar responses of local field potentials (LFPs) to the magnetic stimulation reached layer 6, and the spatiotemporal profiles were very similar to those of the acoustic stimulation, suggesting the activation of the same cortical microcircuit. The horizontal LFP responses to the magnetic stimulation were evoked within a millimeter-wide area around the stimulation coil. The activated cortical area was dependent on the coil orientation, providing useful information on the effective position of the coil relative to the brain surface for modulating cortical circuitry activity. In addition, numerical calculation of the induced electric field in the brain revealed that the inhomogeneity of the horizontal electric field to the surface is critical for micro-coil-induced cortical activation. The results suggest that our micro-coil technique has the potential to be used as a chronic, less-invasive and highly focal neuro-stimulator, and is useful for investigating microcircuit responses to magnetic stimulation for clinical treatment.

Salicylate-induced frequency-map reorganization in four subfields of the mouse auditory cortex.

Salicylate is the active ingredient in aspirin, and in high-doses it is used as an experimental tool to induce transient hearing loss, tinnitus, and hyperacusis. These salicylate-induced perceptual disturbances are associated with tonotopic-map reorganization and neural activity modulation, and such neural correlates have been examined in the central auditory pathway, including the auditory cortex (AC). Although previous studies have reported that salicylate induces increases in noise-burst-evoked neural responses and reorganization of tonotopic maps in the primary AC, little is known about the effects of salicylate on other frequency-organized AC subfields such as the anterior auditory, secondary auditory, and dorsomedial fields. Therefore, to examine salicylate-induced spatiotemporal effects on AC subfields, we measured sound-evoked neural activity in mice before and after the administration of sodium salicylate (SS, 200 mg/kg), using flavoprotein auto-fluorescence imaging. SS-treatment gradually reduced responses driven by tone-bursts with lower (≤8 kHz) and higher (≥25 kHz) frequencies over 3 h, whereas evoked responses to tone-bursts within middle-range frequencies (e.g., 12 and 16 kHz) were sustained and unchanged in the four subfields. Additionally, in each of the four subfields, SS-treatment induced similar reorganization of tonotopic maps, and the response areas selectively driven by the middle-range frequencies were profoundly expanded. Our results indicate that the SS-induced tonotopic map reorganizations in each of the four AC subfields were similar, and only the extent of the activated areas responsive to tone-bursts with specific frequencies was subfield-dependent. Thus, we expect that examining cortical reorganization induced by SS may open the possibility of new treatments aimed at altering cortical reorganization into the normative functional organization.

Transcranial flavoprotein-autofluorescence imaging of sound-evoked responses in the mouse auditory cortex under three types of anesthesia.

The effects of anesthesia on the functional auditory characteristics of cortical neurons, such as spatial and temporal response properties, vary between an anesthetized and an awake subject. However, studies have shown that an appropriate anesthetic method that approaches the awake condition is still useful because of its greater stability and controllability. The present study compared neural response properties from two core fields of the mouse auditory cortex under three anesthetic conditions: urethane; ketamine and xylazine hydrochloride (KX) mixture; and a combination of medetomidine, midazolam, and butorphanol (MMB). To measure sound stimulation in vivo, we recorded flavoprotein-autofluorescent images of endogenous green fluorescence. Under all conditions, fluorescence changes in auditory core subfields in response to tones were observed, and response properties, such as peak intensity, latency, duration, and activated areas were analyzed. Results showed larger response peak intensity, latency, and duration in the core subfields under urethane compared with KX and MMB, with no significant differences between KX and MMB. Conversely, under KX anesthesia the activated areas showed characteristic response properties in a subfield-dependent manner. These results demonstrated the varied effects of anesthesia on response properties in the core subfields of the auditory cortex.