Masaharu Kudoh

Layer‐specific NO dependence of long‐term potentiation and biased NO release in layer V in the rat auditory cortex

1 We investigated the role of nitric oxide (NO) in the induction of long‐term potentiation (LTP) in slices prepared from the rat auditory cortex. 2 Tetanic stimulation of layer IV elicited LTP of field potentials in layer II–III (LTPII–III) and in layer V (LTPV). The magnitude of LTPII–III measured at 30 min after tetanic stimulation was 171 ± 9 % (n= 15, mean ± s.e.m.) of the control measured before tetanic stimulation, while that of LTPV was 138 ± 3 % (n= 17). 3 NO synthase (NOS) inhibitors had no apparent effect on LTPII–III, but LTPV was significantly suppressed (P < 0.001). This suppression of LTPV was significantly antagonized by a NO donor (P < 0.001) or a cGMP analogue (P < 0.001). 4 Small non‐pyramidal neurones in the auditory cortex were stained with an anti‐neuronal NOS antibody. More neurones were stained with the antibody in the deeper cortical layers. 5 We measured neocortical NO release with electrochemical NO probes. Layer IV stimulation elicited significantly more NO release in layer V than in layer II–III (P < 0.001). The amplitude of the increase in NO concentration elicited by stimulation at 20 Hz for 5 s was 380 ± 14 pM (n= 55) in layer V and 55 ± 8 pM (n= 5) in layer II–III. 6 NO release in layer V was partially but significantly suppressed by non‐NMDA (P < 0.002) or NMDA (P < 0.002) receptor antagonists. Simultaneous application of the antagonists of the two types blocked NO release almost completely. 7 These results clearly indicate the NO dependence of the induction of LTPV, and the greater NO release in the deeper layer of the rat auditory cortex.

Delineation of a frequency-organized region isolated from the mouse primary auditory cortex

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.

Multimodal cortical sensory pathways revealed by sequential transcranial electrical stimulation in mice

We investigated polysynaptic cortical pathways linking primary to multimodal sensory association areas in mice using transcranial flavoprotein imaging combined with sequential application of transcranial electrical stimulation (TES). Stimulation of primary visual cortex (V1) elicited activity in lateral and medial areas of secondary visual cortices (V2), which were reciprocally connected. Stimulation of V2 areas elicited activity in area 2. Similarly, corticocortical pathways from primary somatosensory cortex (S1) through the corresponding secondary somatosensory areas (S2) to area 2 were observed. Auditory pathways from primary auditory area (A1) through peripheral region (area 22) to area 2 and from anterior auditory field to area 2 were also found. Stimulation in area 2 elicited activity in part of parietal association cortex (PtA), which was reciprocally connected with area 2, and in some areas near the midline including retrosplenial cortex (RSA). A cortical pathway from RSA through anterior cingulate cortex (aCC) to frontal areas was also visualized. These results indicate that area 2, surrounded by visual, somatosensory and auditory cortices, may receive inputs from all three primary sensory areas, and may send outputs through the parietal association cortex to frontal areas, suggesting that area 2 may have an important role in multimodal sensory integration in mice.

Transcranial electrical stimulation of cortico-cortical connections in anesthetized mice.

We developed a technique of transcranial electrical stimulation (TES) to investigate cortico-cortical connections in mice. After the skull was shaved with the blade of a dental bar, a blunt tip of a needle was gently pushed onto the thinned skull. The skull was deformed by the force, and the subarachnoid space between the skull and the cortex was minimized around the needle tip. Under these conditions, stimulus currents applied to the needle directly flowed into the cortex through the thinned skull. Cortico-cortical functional connections stimulated by this method were visualized by transcranial flavoprotein fluorescence imaging. The cortical responses evoked by TES exhibited spatial and temporal activity patterns comparable to those elicited by a conventional method, in which an electrode is directly inserted into superficial cortical layers. A comparison of the two methods revealed that TES required a slightly stronger stimulus intensity and preferentially activated superficial layers of the cortex compared with the conventional method. Using the new method, we revealed the presence of reciprocal cortico-cortical functional connections between lateral and medial parts of higher visual cortices in mice. This new method combined with transcranial flavoprotein fluorescence imaging allowed us to activate cortico-cortical pathways arising from the primary sensory areas and investigate sensory information flow in the mouse cerebral cortex.

Auditory cortical responses to harmonic type and noise type pup calls in mother rats

foot-shock (Unconditioned Stimulus, US) was investigated by using of optical imaging. Optical signals in the auditory cortex in response to CS (a 12 kHz pure tone) and non-CS (4, 8, 16 kHz pure tones) were recorded before and after normal and sham conditioning. Results showed that the area activated by CS enlarged only after normal conditioning. Furthermore, we report that, after conditioning, auditory information could be retrieved on the basis of an electric foot-shock alone. Before conditioning, the auditory cortex showed no response to a foot-shock presented in the absence of sound. In contrast, after conditioning, the presentation of a foot-shock in the absence of sound elicited activity in the auditory cortex. Additionally, the magnitude of the optical response in the auditory cortex correlated with variation in the electrocardiogram. The area activated in the auditory cortex, in response to the foot-shock, also showed a considerable correspondence to that elicited by the CS sound. Research fund: Grant-in-Aid for Young Scientists (B) 21700435, Grant-in-Aid for Scientific Research on Innovative Areas 21120006.

Neuronal responses to synthetic fricative consonants in the rat auditory fields

foot-shock (Unconditioned Stimulus, US) was investigated by using of optical imaging. Optical signals in the auditory cortex in response to CS (a 12 kHz pure tone) and non-CS (4, 8, 16 kHz pure tones) were recorded before and after normal and sham conditioning. Results showed that the area activated by CS enlarged only after normal conditioning. Furthermore, we report that, after conditioning, auditory information could be retrieved on the basis of an electric foot-shock alone. Before conditioning, the auditory cortex showed no response to a foot-shock presented in the absence of sound. In contrast, after conditioning, the presentation of a foot-shock in the absence of sound elicited activity in the auditory cortex. Additionally, the magnitude of the optical response in the auditory cortex correlated with variation in the electrocardiogram. The area activated in the auditory cortex, in response to the foot-shock, also showed a considerable correspondence to that elicited by the CS sound. Research fund: Grant-in-Aid for Young Scientists (B) 21700435, Grant-in-Aid for Scientific Research on Innovative Areas 21120006.

Zic2 plays an essential role in the formation of auditory neural circuit

Zic2 is a causal gene of holoprosencephaly (a dysgenesis of medial forebrain). Although it is broadly expressed in CNS, there was a difficulty to fully show its role due to its critical role in early embryogenesis. Here we developed a conditionally targeted Zic2 mutant mice and clarified its role in the development of dorsal cochlear nucleus (DCoN) and in the auditory function of mature mice. Soon after the neural tube closure, Zic2 and its close relatives (Zic1 and Zic3) are differentially expressed in hindbrain region along the rostrocaudal axis. Zic2 expression was dominant in the DCoN forming region. In Zic2 hypomorphic mutants (60% of wild type level), slight reduction of DCoN size was observed whereas the DCoN size reduction was severe in the midbrainhindbrain restricted Zic2 conditional knockout (CKO). Both granule cells and unipolar brush cells were decreased in DCoN. We observed the increased acoustic startle response and the altered auditory brain stem responses in both Zic2 hypomorphic and Zic2 CKO animals. Furthermore, we measured the activities of the primary auditory cortices during various sound stimuli application by means of autofluorescence imaging. These results indicated that optimal Zic2 gene dosage is a critical parameter for the auditory neural circuit formation and the auditory function. Further analyses using the Zic2 mutants would be beneficial for understanding physiological regulation of auditory information processing in mammalian. Research fund: RIKEN BSI funds.

Two parallel pathways for sound discrimination in the rat auditory cortex

s / Neuroscience Research 68S (2010) e223–e334 e273 P2-i10 Developmental and gender differences in nicotine responsiveness of auditory cortical responses Hideki Kawai 1,2 , Ho-An Kang 2, Ronit Lazar 2, Raju Metherate 2 1 Dept Bioinformatics, Soka Univ, Tokyo 2 Dept of Neurobiology & Behavior, Univ of California, Irvine, USA Smoking during adolescence increases the risk of tobacco addiction, and nicotinic modulation of sensory-cognitive function could play a role in establishing dependence. Although nicotine enhances sensory-evoked responses in adults, effects during adolescence are unknown. Here, we determined whether nicotinic alteration of auditory cortical processing in mice depends on age (postnatal day (P) 21–90) and gender. We examined tone (characteristic frequency, CF) -evoked current source density (CSD) profiles in auditory cortex using a 16-channel multiprobe, and determined effects of systemic nicotine (0.7 mg/kg, free base). Nicotine increased tone-evoked Input (latency <5 ms, presumed thalamocortical input) and Late Intracortical (30–100 ms) CSD sinks in adolescent mice more than in younger mice or adults. The increased responsiveness peaked earlier in females (P26–30) than in males (P36–40). Enhancement of Early Intracortical (5–20 ms) sinks increased gradually over development in males, but peaked during early adolescence in females. At ages of peak nicotine responsiveness, we examined effects of antagonists injected intracortically. Dihydro-erythroidine, an antagonist specific to receptors containing 4 and 2 subunits ( 4 2nAChRs), blocked the effects of nicotine on Input and Early Intracortical responses. Late Intracortical enhancement was blocked in females only. Methyllycaconitine, an antagonist specific to nAChRs containing 7 subunits ( 7-nAChRs), blocked enhancement of Input responses in males and Early Intracortical responses in females. These data indicate that nicotinic enhancement of sensory evoked responses in cortex peaks during adolescence in a gender-dependent manner. doi:10.1016/j.neures.2010.07.1213 P2-i11 Two parallel pathways for sound discrimination in the rat auditory cortex Go Ogawa , Masaharu Kudoh Dept Physiol, Teikyo Univ Sch Med, Tokyo, Japan We have reported that discrimination learning of synthetic vowels with multiple formants was impaired by bilateral lesions of rostral or dorsal parts of the auditory cortex in rats, while lesions of the primary auditory cortex (AI) had no clear effect. Flavoprotein autofluorescence imaging shows that synthetic formants or vowels evoke prominent responses in the anterior (AAF) and dorsal (DAF) auditory field, which corresponds to the sites of rostral and dorsal AC lesions, in naïve and discrimination achieved rats. These results indicate that the AAF and DAF play a critical role in discrimination learning of vowels in rats. On the other hand, pure tones evoked clear fluorescence responses in the AI and ventral auditory field (VAF). In the present study, we investigated functional connections of auditory fields by electrical stimulation (negative–positive currents 100 s, 300 A, 100 Hz, 100 pulses). Electrical stimulation applied into AAF elicited fluorescence responses in DAF and ventral part of AAF (anteroventral auditory field), while that into AI evoked responses in VAF and anteroventral auditory fields. These findings suggest that there are two parallel pathways, AAF to DAF pathway and AI to VAF pathway, which have different functions for sound discrimination in the rat auditory cortex. doi:10.1016/j.neures.2010.07.1214 P2-i12 Dynamic population coding of auditory information in mammal’s cortex Yoshiki Kashimori 1 , Youichi Suzukawa 2 1 Department of Engineering Science, University of Electro-Communications 2 Graduate School of Information Systems Animals utilize auditory information for survival and communications of conspecifics. A sequence of sound is analyzed in animals’brain as elementary components such as notes and syllables. It has been reported that auditory information is represented by spatiotemporal activity of primary auditory cortex. However, how the elementary components of sound are encoded from the spatiotemporal activity of neurons is poorly understood. To address this issue, we present a model of auditory cortex, which performs a hierarchical processing of auditory information. The model consists of three layers of 2-dimensional networks. The first layer is a model of primary auditory cortex, in which the auditory information is represented as a spatiotemporal activity of cortical neurons. The second layer consists of feature-detective neurons, which encode the information of the spatiotemporal correlations between notes. The third layer combines the information of notes encoded by the second layer and represents a word as a dynamical attractor. Using this model, we show that the aspects of the spatiotemporal activity in the primary cortex are encoded by a combination of feature-detective neurons and then by a dynamical attractor in higher-order cortex. The extraction of spatiotemporal correlations between notes in the second layer is needed to make dynamical attractor in the third layer. The present study provides a clue for understanding the mechanism of how the information of notes and syllables are constructed from spatiotemporal activity of the primary auditory cortex. doi:10.1016/j.neures.2010.07.1215 P2-i13 Neural correlates of auditory stream segregation –Stimulus-onset synchrony within the auditory cortex– Takahiro Noda , Hirokazu Takahashi, Ryohei Kanzaki Graduate School of Information Science and Technology, MechanoInformatics, Kanzaki & Takahashi Lab, University of Tokyo Auditory system can reconstruct sequential sounds into several perceptual groups. To form perceptual objects from acoustic scene is called auditory stream segregation. In psychophysics, alternating tone sequences differing in frequency (ABA-ABA-...) have been studied as a simple analysis object. It is noted that frequency difference ( F) and inter tone interval (ITI) between A-tone and B-tone affect segregation, i.e., separation of streams from alternating tone sequences. The physiological correlates of the segregation in the auditory cortex are likely due to frequency selectivity and forward masking. However, the time–frequency filter property of auditory cortex could only partly explain the perceptual boundary of the stream segregation. It is hardly discussed how far such intrinsic properties of auditory cortex account for the psychophysical findings. The present study focuses on neural synchrony in auditory cortex, which has specific characteristics under conditions inducing stream in psychophysics. We recorded Local Field Potentials in the auditory cortices of anesthetized rats using microelectrode arrays and analyzed phase synchrony to the tone onset across auditory field. At a local maximum of Atone evoked responses (A-tone local focus), the phase of tone onset became similar to that of the segregated tone sequences when Fs were large and ITIs loosely matched the period of oscillation. On the contrary, synchrony across auditory fields was less sensitive to ITIs and Fs. However, synchrony in the delta-band (1–4 Hz) tended to be similar to that of the segregated sequences under shorter ITIs and larger Fs. While the tendency of phase at A-tone local focus represents filter property emphasizing acoustic differences, the trends of synchrony across auditory fields were more consistent with the psychophysical findings. These results suggest that spatial entrainment of neuronal ensemble activity is the inherent mechanism of the streaming in the auditory cortex. doi:10.1016/j.neures.2010.07.1216 P2-i14 Neural origin of mismatch negativity-like response in rat auditory cortex Tomoyo Isoguchi 1,2 , Ryohei Kanzaki 1,2, Hirokazu Takahashi 1,2,3 1 Department of Mechano-Informatics Graduate School of Information Science and Technology, University of Tokyo 2 Research Center for Advanced Science and Technology, University of Tokyo 3 PRESTO, JST Purpose: Auditory Mismatch Negativity (MMN) is a long-latency, negative deflection in the evoked potential in response to a change in some features of repeated stimuli. In the past EEG and MEG studies, the spatial distribution of MMN has not been satisfactorily characterized because of the low spatial resolution of the measurement. On the other hand, MMN-like responses have been densely mapped in some animal models, yet there are some debates on whether these responses have the same origin as the human MMN. In this study, we attempted to establish a rodent model to investigate MMN. First, we densely mapped MMN-like responses and the middle latency potential (P1) in the rat auditory cortex using a surface microelectrode array. Second, we tested whether the MMN-like response is mediated by NMDA receptors along with human MMN. Methods: We used wistar rats (9 weeks old, 270–280 g) for the experiment. A surface microelectrode array was mounted on the exposed right auditory cortex of the anesthetized rats. The oddball situation induced MMN-like responses. We then applied AP5 by putting an agarose gel seat (1%) including AP5 (100 M) on the surface of auditory cortex for 10 minutes. After the application of AP5, we mapped and quantified MMN-like responses again.

Converged inputs to multimodal association cortices from the primary sensory areas revealed by sequential transcranial electrical stimulation in mice

Brachial plexus (BP) injury is sometimes repaired by nerve crossing for bypassing the injured sites. Successful functional recovery after such operation suggests the presence of some plasticity after nerve crossing. To test this hypothesis, we investigated somatosensory cortical responses after nerve crossing in mice using transcranial flavoprotein fluorescence imaging. Vibratory stimuli applied to the left forepaw elicited bilateral cortical responses. Photo-inactivation of the left cortex suppressed the left and right responses, indicating involvement of callosal fibers for producing the right cortical responses. The right cortical responses after the nerve crossing were reduced in cortex-specific, heterotypic NR1 knockout mice, indicating that experience-dependent plasticity in inter-hemispheric pathways has an important role for functional recovery after nerve crossing in patients with BP injury.

Roles of glutamate receptors in the induction of input-specific long-term depression in the rat auditory cortex

To express recombinant NMDA receptor channels in CNS neurons, we constructed three recombinant adenoviruses encoding NRl, NRl(N598R) and NRBB, respectively. In NRl(N598R), asparagine in N-site of the wild type NRl was replaced with arginine (R) by site-directed mutagenesis. Using the whole-cell patch clamp technique, we examined functional expressions of heteromeric NRl/NRZB and NRl(N598R)/NR2B receptors in PC12 cells co-infected with the adenoviruses. NRVNRZB receptors thus expressed were permeable to Ca” and blocked by Mg*‘, whereas NRl(N598R)/NR2B receptors were Ca 2’-impermeable and insensitive to Mg*+. Next, we infected cultured rat hippocampal neurons with the adenoviruses bearing NRl(N598R) and NRZB. Both Ca2’ permeability and Mg*+ block in NMDA receptors were markedly reduced. The slow EPSC mediated by NMDA receptors also became less sensitive to Mg*‘. We conclude that the NRl(N598R)/NR2B receptors introduced by the adenoviral vectors function as postsynaptic receptors.