Molecular diversity of clustered protocadherin-α required for sensory integration and short-term memory in mice.

Clustered protocadherins (Pcdhs) are neuronal cell adhesion molecules characterized by homophilic adhesion between the tetramers of 58 distinct isoforms in mice. The diversity of Pcdhs and resulting highly-specific neuronal adhesion may be required for the formation of neural circuits for executing higher brain functions. However, this hypothesis remains to be tested, because knockout of Pcdh genes produces abnormalities that may interfere with higher brain functions indirectly. In Pcdh-α1,12 mice, only α1, α12 and two constitutive isoforms are expressed out of 14 isoforms. The appearance and behavior of Pcdh-α1,12 mice are similar to those of wild-type mice, and most abnormalities reported in Pcdh-α knockout mice are not present in Pcdh-α1,12 mice. We examined Pcdh-α1,12 mice in detail, and found that cortical depression induced by sensory mismatches between vision and whisker sensation in the visual cortex was impaired. Since Pcdh-α is densely distributed over the cerebral cortex, various types of higher function are likely impaired in Pcdh-α1,12 mice. As expected, visual short-term memory of space/shape was impaired in behavioral experiments using space/shape cues. Furthermore, behavioral learning based on audio-visual associative memory was also impaired. These results indicate that the molecular diversity of Pcdh-α plays essential roles for sensory integration and short-term memory.

Brain-computer interface training combined with transcranial direct current stimulation in patients with chronic severe hemiparesis: Proof of concept study.

OBJECTIVE: Brain-computer interface technology has been applied to stroke patients to improve their motor function. Event-related desynchronization during motor imagery, which is used as a brain-computer interface trigger, is sometimes difficult to detect in stroke patients. Anodal transcranial direct current stimulation (tDCS) is known to increase event-related desynchronization. This study investigated the adjunctive effect of anodal tDCS for brain-computer interface training in patients with severe hemiparesis. SUBJECTS: Eighteen patients with chronic stroke. DESIGN: A non-randomized controlled study. METHODS: Subjects were divided between a brain-computer interface group and a tDCS- brain-computer interface group and participated in a 10-day brain-computer interface training. Event-related desynchronization was detected in the affected hemisphere during motor imagery of the affected fingers. The tDCS-brain-computer interface group received anodal tDCS before brain-computer interface training. Event-related desynchronization was evaluated before and after the intervention. The Fugl-Meyer Assessment upper extremity motor score (FM-U) was assessed before, immediately after, and 3 months after, the intervention. RESULTS: Event-related desynchronization was significantly increased in the tDCS- brain-computer interface group. The FM-U was significantly increased in both groups. The FM-U improvement was maintained at 3 months in the tDCS-brain-computer interface group. CONCLUSION: Anodal tDCS can be a conditioning tool for brain-computer interface training in patients with severe hemiparetic stroke.

Transcranial direct current stimulation enhances mu rhythm desynchronization during motor imagery that depends on handedness.

Transcranial direct current stimulation (tDCS) can modulate the amplitude of event-related desynchronization (ERD) that appears on the electroencephalogram (EEG) during motor imagery. To study the effect of handedness on the modulating effect of tDCS, we compared the difference in tDCS-boosted ERD during dominant and non-dominant hand motor imagery. EEGs were recorded over the left sensorimotor cortex of seven healthy right-handed volunteers, and we measured ERD induced either by dominant or non-dominant hand motor imagery. Ten minutes of anodal tDCS was then used to increase the cortical excitability of the contralateral primary motor cortex (M1), and ERD was measured again. With anodal tDCS, we observed only a small increase in ERD during non-dominant hand motor imagery, whereas the same stimulation induced a prominent increase in ERD during dominant hand motor imagery. This trend was most obvious in the participants who used their dominant hand more frequently. Although our study is preliminary because of a small sample size, these results suggest that the increase in ERD by applying anodal tDCS was stronger on the dominant side than on the non-dominant side. The background excitability of M1 may determine the strength of the effect of anodal tDCS on ERD by hand motor imagery.

Timing-dependent effects of whisker trimming in thalamocortical slices including the mouse barrel cortex.

Whisker trimming produces depression of cortical responses in the barrel cortex. However, it is unclear how the developmental timing modifies the effects of whisker trimming. We investigated cortical responses in thalamocortical slices that included the mouse barrel cortex using flavoprotein fluorescence imaging. A topological relationship was observed between the thalamic stimulated sites and cortical areas showing fluorescence changes. By adjusting the position of the thalamic stimulated sites and the cortical windows in which amplitudes of the fluorescence changes were measured, we succeeded to reduce the variability of cortical responses between slices. We then investigated the effects of whisker trimming in the thalamocortical slices. Whisker trimming from 4 weeks to 8 weeks (at 4-8 weeks) of age significantly reduced cortical responses at 8 weeks. However, whisker trimming started before 4 weeks produced only slight depression or no significant effect on the thalamocortical responses. As sensory deprivation during a critical developmental period is known to prevent elimination of synapses, the presence of aberrant synapses may compensate the cortical depression induced by whisker trimming started before 4 weeks. To test this possibility, whisker trimming performed at 0-6 or 0-7 weeks of age was followed by regrowth of whiskers for 1-2 weeks. Clear and significant potentiation of cortical responses was observed in these mice at 8 weeks when compared with those of naive mice of the same age. Overall, these data suggest that whisker trimming, producing depression of thalamocortical responses, prevents elimination of aberrant synapses during a critical developmental period before 4 weeks in the mouse barrel 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.

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.

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.

Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human neuronal nitric-oxide synthase and inducible nitric-oxide synthase flavin domains.

Neuronal nitric-oxide synthase (nNOS) differs from inducible NOS (iNOS) in both its dependence on the intracellular Ca2+ concentration and the production rate of NO. To investigate what difference(s) exist between the two NOS flavin domains at the electron transfer level, we isolated the recombinant human NOS flavin domains, which were co-expressed with human calmodulin (CaM). The flavin semiquinones, FADH* and FMNH*, in both NOSs participate in the regulation of one-electron transfer within the flavin domain. Each semiquinone can be identified by a characteristic absorption peak at 520 nm (Guan, Z.-W., and Iyanagi, T. (2003) Arch. Biochem. Biophys. 412, 65-76). NADPH reduction of the FAD and FMN redox centers by the CaM-bound flavin domains was studied by stopped-flow and rapid scan spectrometry. Reduction of the air-stable semiquinone (FAD-FMNH*) of both domains with NADPH showed that the extent of conversion of FADH2/FMNH* to FADH*/FMNH2 in the iNOS flavin domain was greater than that of the nNOS flavin domain. The reduction of both oxidized domains (FAD-FMN) with NADPH resulted in the initial formation of a small amount of disemiquinone, which then decayed. The rate of intramolecular electron transfer between the two flavins in the iNOS flavin domain was faster than that of the nNOS flavin domain. In addition, the formation of a mixture of the two- and four-electron-reduced states in the presence of excess NADPH was different for the two NOS flavin domains. The data indicate a more favorable formation of the active intermediate FMNH2 in the iNOS flavin domain.