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.

Transcranial photo-inactivation of neural activities in the mouse auditory cortex.

Flavoprotein fluorescence in the brain is intimately coupled with neuronal aerobic energy metabolism. If flavoproteins are photobleached, neural activities may be affected owing to dysfunction in aerobic energy metabolism in mitochondria. We tested this possibility in cortical slices from mice, and found that exposure to blue light (lambda = 475 nm) derived from a 20 mW diode laser for 50 min suppresses trans-synaptic components of field potentials. This finding formed the basis of a transcranial photo-inactivation technique, that was used to investigate auditory signal transmission between the anterior auditory field (AAF) and the primary auditory cortex (AI) in anesthetized mice. Cortical responses in AAF and AI, elicited by 5 kHz tonal stimuli, were visualized using transcranial flavoprotein fluorescence imaging. After determining responsive areas in AAF and AI, the auditory cortex was exposed to the blue diode laser via the intact skull, while either AAF or AI was protected with a piece of carbon paper. Although the photo-inactivation of AI had no significant effect on the fluorescence responses in AAF, the photo-inactivation of AAF significantly reduced the fluorescence responses in AI, indicating the presence of auditory signal transmission from AAF to AI.

Long-term depression induced by local tetanic stimulation in the rat auditory cortex.

In sensory cortices, synaptic plasticities such as long-term potentiation (LTP) and long-term depression (LTD) have important roles in the development of neural circuits and sensory information processing. However, the differential roles and mechanisms of the various types of LTP and LTD are not clear. In the present study, we investigated LTP and two types of LTD in slices obtained from the rat auditory cortex. Supragranular field potentials elicited by layer VI stimulation were recorded through a metal electrode. Transsynaptic field potentials exhibited marked LTP after tetanic stimulation (TS, 100 Hz for 1 s) was applied to layer VI. The same field potential components exhibited LTD after low-frequency stimulation (LFS, 1 Hz for 900 s) was applied to layer VI. LTD of supragranular field potentials was also induced by local TS applied to supragranular layers 0.3 mm from the recording site. Neither LTP nor LTD of either type was induced in the presence of 50 muM d-(-)-2-amino-5-phosphonovalerate (APV), an NMDA receptor antagonist. However, 500 muM (+)-alpha-methyl-4-carboxyphenylglycine (MCPG), an antagonist of metabotropic glutamate receptors, had no effect. LTD induced by LFS and that induced by local TS were suppressed in the presence of 3 muM bicuculline, an antagonist of GABA(A) receptors. Each of these forms of LTD occluded the other. These results and intracellular recordings in supragranular pyramidal neurons during LFS and local TS strongly suggest that the two types of LTD share common neural circuits for their induction.

Roles of nitric oxide as a vasodilator in neurovascular coupling of mouse somatosensory cortex.

Neural activities trigger regional vasodilation in the brain. Diffusible messengers such as nitric oxide (NO) and prostanoids are considered to work as vasodilators in neurovascular coupling. However, their roles are still controversial. In the present study, cortical images of neural activities and vasodilation were recorded through the intact skull of C57BL/6 mice anesthetized with urethane. Flavoprotein fluorescence responses elicited by vibratory hindpaw stimulation were followed by darkening of arteriole images reflecting vasodilation in the somatosensory cortex. Vasodilation was also observed in light reflection images at the wavelength of 570 nm in the same mice. We perfused the surface of the cortex under the skull with 100 microM N(G)-nitro-l-arginine (l-NA), an inhibitor of NO synthase (NOS), and 10 microM indomethacin, an inhibitor of cyclooxygenase (COX). These drugs suppressed vasodilation without changing flavoprotein fluorescence responses. A mixture of l-NA and indomethacin almost completely eliminated vasodilation. In mice lacking neuronal NOS (nNOS), activity-dependent vasodilation was significantly suppressed compared with that in littermate control mice, while that in mice lacking cytosolic phospholipase A2 alpha (cPLA2alpha) was unchanged. These results indicate that NO works as a vasodilator in neurovascular coupling of the mouse somatosensory cortex.

Experience-dependent formation of activity propagation patterns at the somatosensory S1 and S2 boundary in rat cortical slices.

Somatosensory information is serially processed by the primary (S1) and secondary (S2) cortices, which can be identified in fresh cortical slices. We visualized activity propagation between S1 and S2 in rat cortical slices using flavoprotein fluorescence imaging. When S1 was stimulated, fluorescence responses extended into S2, while responses hardly propagated to S1 following S2 stimulation. The dominant activity propagation pattern from S1 to S2 was not affected by antagonists of glutamate or GABA(A) receptors. Ca(2+) imaging and electrophysiological recordings confirmed the anisotropic activity propagation pattern. This pattern could be formed as a result of serial information processing in S1 and S2. To test this hypothesis, activity propagation was investigated in cortical slices prepared 2 weeks or 3 days after trimming contralateral whiskers that provide massive inputs to S1. Supragranular activities in the barrel cortex were clearly suppressed. Furthermore, activities elicited in the rostral small vibrissae/mouth area of S1 near the border between S1 and S2 spread into the adjacent barrel cortex rather than into S2. Behavioral effects of whisker trimming were evaluated using a test, in which rats chose one of two bridges that had a wall on the right or left side only. Immediately after hemilateral whisker trimming, rats preferred to use the bridge with a wall close to the intact side. However, this preference disappeared 3 days after trimming. Modified activities observed in cortical slices after whisker trimming might be mechanisms for this behavioral compensation. These findings suggest experience-dependent formation of activity propagation patterns in the somatosensory cortex.

Functional local connections with differential activity-dependence and critical periods surrounding the primary auditory cortex in rat cerebral slices.

Sensory information is processed in neural networks connecting the primary sensory cortices with surrounding higher areas. Here, we investigated the properties of local connections between the primary auditory cortex (area 41) and surrounding areas (areas 20, 36, 18a and 39) in rat cerebral slices. Neural activities elicited by repetitive electrical stimulation were visualized using the activity-dependent changes in endogenous fluorescence derived from mitochondrial flavoproteins, which mostly reflect activities produced by polysynaptic glutamatergic transmission. Polysynaptic feedforward propagation was dominant compared with the corresponding polysynaptic feedback propagation between the primary (area 41) and secondary (areas 20 and 36) auditory cortices, while such a tendency was less clear in other pathways. Long inter-areal (>1 mm) propagation with the same dominancy was observed after layer V stimulation between areas 41 and 20, and was not affected by cutting the underlying white matter. Activity-dependent changes in neural activities induced by low-frequency stimulation in the presence of 1 microM bicuculline were investigated using Ca2+ imaging. Significant potentiation of the polysynaptic Ca2+ activities was only observed in polysynaptic feedforward pathways from the primary to secondary auditory cortices. Experience-dependence of the connections between areas 41 and 20 was investigated using flavoprotein fluorescence imaging. The activities from areas 41 to 20 were reduced by cochlear lesions produced at P12 but not at P28, while the activities from areas 20 to 41 were reduced by the lesions at P28, suggesting the critical period for the polysynaptic feedforward connection was before P28, while for the polysynaptic feedback connection was after P28.

Sound sequence discrimination learning motivated by reward requires dopaminergic D2 receptor activation in the rat auditory cortex.

We have previously reported that sound sequence discrimination learning requires cholinergic inputs to the auditory cortex (AC) in rats. In that study, reward was used for motivating discrimination behavior in rats. Therefore, dopaminergic inputs mediating reward signals may have an important role in the learning. We tested the possibility in the present study. Rats were trained to discriminate sequences of two sound components, and licking behavior in response to one of the two sequences was rewarded with water. To identify the dopaminergic inputs responsible for the learning, dopaminergic afferents to the AC were lesioned with local injection of 6-hydroxydopamine (6-OHDA). The injection attenuated sound sequence discrimination learning, while it had no effect on discrimination between the sound components of the sequence stimuli. Local injection of 6-OHDA into the nucleus accumbens attenuated sound discrimination learning. However, not only discrimination learning of sound sequence but also that of the sound components were impaired. SCH23390 (0.2 mg/kg, i.p.), a D1 receptor antagonist, had no effect on sound sequence discrimination learning, while it attenuated the licking behavior to unfamiliar stimuli. Haloperidol (0.5 mg/kg, i.p.), a D2 family antagonist, attenuated sound sequence discrimination learning, while it had no clear suppressive effect on discrimination of two different sound components and licking. These results suggest that D2 family receptors activated by dopaminergic inputs to the AC are required for sound sequence discrimination learning.

Requirement of the auditory association cortex for discrimination of vowel-like sounds in rats.

We investigated the roles of the auditory cortex in discrimination learning of vowel-like sounds consisting of multiple formants. Rats were trained to discriminate between synthetic sounds with four formants. Bilateral electrolytic lesions including the primary auditory cortex and the dorsal auditory association cortex impaired multiformant discrimination, whereas they did not significantly affect discrimination between sounds with a single formant or between pure tones. Local lesions restricted to the dorsal/rostral auditory association cortex were sufficient to attenuate multiformant discrimination learning, and lesions restricted to the primary auditory cortex had no significant effects. These findings indicate that the dorsal/rostral auditory association cortex but not the primary auditory cortex is required for discrimination learning of vowel-like sounds with multiple formants in rats.

Enduring critical period plasticity visualized by transcranial flavoprotein imaging in mouse primary visual cortex.

Experience-dependent plasticity in the visual cortex was investigated using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. On- and off-responses in the primary visual cortex were elicited by visual stimuli. Fluorescence responses and field potentials elicited by grating patterns decreased similarly as contrasts of visual stimuli were reduced. Fluorescence responses also decreased as spatial frequency of grating stimuli increased. Compared with intrinsic signal imaging in the same mice, fluorescence imaging showed faster responses with approximately 10 times larger signal changes. Retinotopic maps in the primary visual cortex and area LM were constructed using fluorescence imaging. After monocular deprivation (MD) of 4 d starting from postnatal day 28 (P28), deprived eye responses were suppressed compared with nondeprived eye responses in the binocular zone but not in the monocular zone. Imaging faithfully recapitulated a critical period for plasticity with maximal effects of MD observed around P28 and not in adulthood even under urethane anesthesia. Visual responses were compared before and after MD in the same mice, in which the skull was covered with clear acrylic dental resin. Deprived eye responses decreased after MD, whereas nondeprived eye responses increased. Effects of MD during a critical period were tested 2 weeks after reopening of the deprived eye. Significant ocular dominance plasticity was observed in responses elicited by moving grating patterns, but no long-lasting effect was found in visual responses elicited by light-emitting diode light stimuli. The present results indicate that transcranial flavoprotein fluorescence imaging is a powerful tool for investigating experience-dependent plasticity in the mouse visual cortex.

Roles of the auditory cortex in discrimination learning by rats.

We investigated the roles of the auditory cortex in sound discrimination learning in Wistar rats. Absolute pitch or relative pitch can be used as discrimination cues in sound frequency discrimination. To clarify this, rats were trained to discriminate between rewarded (S+) and unrewarded (S-) test stimuli (S+ frequency>S- frequency). After learning was acquired by rats, performance was tested in a new test in which S+ frequency was constant but S+ frequencyS- frequency but both frequencies were increased. If the discrimination cue of the first test was preserved in the new test, performance following change of testing procedures was expected to remain high. The measured performance suggested that rats used relative pitch in half octave discrimination (difference between S+ and S- frequencies, 0.5 octave), and absolute pitch in octave discrimination (difference between S+ and S- frequencies, 1.0 octave). Bilateral lesions in the auditory cortex had almost no effect on performance before procedure change. Furthermore, performance following procedure change was not affected by lesions in the auditory cortex when the discrimination cue was preserved. However, performance was impaired by lesions in the auditory cortex when a new discrimination cue was used following procedure change. Lesions in the auditory cortex also impaired multimodal discrimination between sound and sound plus light. The present findings suggest that the auditory cortex plays a role as a sensory interface of the higher cortices required for flexible learning and multimodal discrimination.

Transcranial fluorescence imaging of auditory cortical plasticity regulated by acoustic environments 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.

Transcranial imaging of audiogenic epileptic foci in the cortex of DBA/2J mice.

Exposure to intense sound stimuli induces audiogenic seizures in DBA/2J mice. We investigated cortical activities during sound stimulation using flavoprotein fluorescence imaging. Most DBA/2J mice had seizures during intense sound stimulation, with more than half surviving after seizures. Surviving mice were anesthetized with urethane (1.6 g/kg, intraperitoneal), and the skull was exposed and then covered with clear resin. More than 3 days after surgery, the mice were lightly anesthetized with urethane (0.8 g/kg) and cortical activities during intense sound stimulation were visualized. Focal responses appeared near the somatosensory cortex together with spike activities localized in the response area. These findings indicate that epileptic foci of audiogenic seizure are formed in the cortex of DBA/2J mice.

Endogenous fluorescence imaging of somatosensory cortical activities after discrimination learning in rats.

Aerobic energy metabolism in the brain is reflected as changes in the green fluorescence of mitochondrial flavoproteins, and the activity-dependent changes in endogenous fluorescence are applicable for functional brain imaging. To understand the roles of cortical plasticity in discrimination learning, we used flavoprotein fluorescence imaging to visualize changes of neural activities in the rat primary somatosensory cortex (SI) after learning. Rats were trained to discriminate floor vibration at rewarded and unrewarded frequencies. After this discrimination learning was accomplished in 3-5 days, the rats were anesthetized with urethane (1.5 g/kg, i.p.), and neural responses were recorded in SI during flutter stimuli applied to the contralateral hindpaw. The fluorescence responses to the stimuli at unrewarded frequencies were selectively depressed in the trained rats, which had behaviorally neglected unrewarded stimuli. The depression of cortical responses was not observed in the rats trained with rewarded stimuli only. Therefore, the stimulus-specific depression in SI might explain a part of neural mechanisms underlying discrimination behavior. To reproduce the stimulus-specific depression of cortical responses in anesthetized rats, tetanic cortical stimulation was paired with flutter stimulation applied to the hindpaw. Selective depression of fluorescence responses or field potentials in SI was induced by the paired stimulation. Our findings suggest that some intracortical circuits in SI are specifically tuned to and modulated by unrewarded stimuli of a particular frequency while SI neurons are responsive to both of rewarded and unrewarded stimuli. The present results indicate the usefulness of flavoprotein fluorescence imaging for investigating somatosensory cortical plasticity after learning.

Characteristics of sound discrimination enhancement after sound exposure in adult rats.

The auditory perceptual capacity of adult rats was assessed with regard to performance during 2-sound-discriminative operant conditioning. If the animals were passively exposed to a sound stimulus before the conditioning, performance was improved (sound-exposure-enhanced discrimination [SED]). SED had a stimulus specificity that could not be predicted in terms of the cochleotopy. SED was obstructed by D-2-amino-5-phosphonovaleric acid, an antagonist of N-methyl-D-aspartate receptors, infused into the auditory cortex (AC). No evidence supported the hypothesis that SED was due to a change of attention or motivation or to interference in the association process (e.g., latent inhibition). These findings suggest that passive auditory experience can enhance the perceptual capacity of an adult rat's AC.

Sound sequence discrimination learning is dependent on cholinergic inputs to the rat auditory cortex.

In rat auditory cortex (AC) slices, synaptic potentiation following heterosynaptic stimulation is affected by the stimulus sequence used for induction. It was hypothesized that this sequence-dependent plasticity might be partly involved in the cellular mechanisms underlying sound sequence discrimination. Sequence dependence is abolished by muscarinic receptor antagonists. Therefore, dependence of sound sequence discrimination learning on cholinergic inputs to the rat AC was investigated. Rats were trained to discriminate the sequences of two sound components and a licking behavior in response to one of two possible sequences was rewarded with water. Atropine, a muscarinic receptor antagonist, attenuated sound sequence discrimination learning. The acquired sound sequence discrimination was not affected by atropine. Injections of the cholinergic immunotoxin 192IgG-saporin into the AC suppressed sound sequence discrimination learning, while discrimination between the two sound components was not affected. An inhibitor of M-current, linopirdine, restores the sequence dependence of synaptic potentiation in the AC slices suppressed by atropine. In this study, sound sequence discrimination learning attenuated by 192IgG-saporin was also restored by linopirdine. These similarities between sequence dependent plasticity in the AC slices and sound sequence discrimination learning support the hypothesis that the former is involved in the cellular mechanisms underlying the latter.

Short-term plasticity visualized with flavoprotein autofluorescence in the somatosensory cortex of anaesthetized rats.

In the present study, short-term plasticity of somatosensory neural responses was investigated using flavoprotein autofluorescence imaging in rats anaesthetized with urethane (1.5 g/kg, i.p.) Somatosensory neural activity was elicited by vibratory skin stimulation (50 Hz for 1 s) applied on the surface of the left plantar hindpaw. Changes in green autofluorescence (lambda = 500-550 nm) in blue light (lambda = 450-490 nm) were elicited in the right somatosensory cortex. The normalised maximal fluorescence responses (deltaF/F) was 2.0 +/- 0.1% (n = 40). After tetanic cortical stimulation (TS), applied at a depth of 1.5-2.0 mm from the cortical surface, the responses elicited by peripheral stimulation were significantly potentiated in both peak amplitude and size of the responsive area (both P < 0.02; Wilcoxon signed rank test). This potentiation was clearly observed in the recording session started 5 min after the cessation of TS, and returned to the control level within 30 min. However, depression of the responses was observed after TS applied at a depth of 0.5 mm. TS-induced changes in supragranular field potentials in cortical slices showed a similar dependence on the depth of the stimulated sites. When TS was applied on the ipsilateral somatosensory cortex, marked potentiation of the ipsilateral responses and slight potentiation of the contralateral responses to peripheral stimulation were observed after TS, suggesting the involvement of commissural fibers in the changes in the somatosensory brain maps. The present study clearly demonstrates that functional brain imaging using flavoprotein autofluorescence is a useful technique for investigating neural plasticity in vivo.

Activity-dependent persisting modification of polysynaptic neural circuits involving layer V pyramidal neurons in rat auditory cortex in vitro.

Synaptic plasticity in polysynaptic neural circuits permits modulation of the dynamic properties of these circuits. We investigated the properties of polysynaptic potentiation in pyramidal neurons in layer V of rat auditory cortex (AC) slices using the perforated patch clamp technique. The GABAA receptor inhibitor bicuculline was used to facilitate polysynaptic activity. The amplitude and duration of the polysynaptic activity were both gradually potentiated with repetitive stimulation (RS) at 12 s intervals. Potentiation was saturated within 10 min of the onset of RS. After the cessation of RS, the polysynaptic responses returned to control levels within 30 min. RS-induced potentiation was confirmed by fluorescence imaging of slices loaded with the Ca2+ indicator rhod-2. Such potentiation was not induced by stimulation at 60 s intervals. The magnitude of the RS-induced potentiation in layer V pyramidal neurons in the AC was greater than that in either layer II/III pyramidal neurons in the AC or layer V pyramidal neurons in the visual cortex. The NMDA receptor antagonist APV (100 microm), inhibited RS-induced potentiation. When stimulated at 1 Hz, the potentiated response appeared rapidly. In the absence of bicuculline, RS consisting of five pulses at 30 ms intervals, repeated at 12 s intervals for 10 min, elicited potentiation of firing activity, suggesting that the potentiation is independent of bicuculline. The present study demonstrates the dynamic properties of polysynaptic circuits involving layer V pyramidal neurons in the AC are strongly affected by activity-dependent synaptic potentiation.