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It is important to study the neural connectivities and functions in primates. For this purpose, it is critical to be able to transfer genes to certain neurons in the primate brain so that we can image the neuronal signals and analyze the function of the transferred gene. Toward this end, our team has been developing gene transfer systems using viral vectors. In this review, we summarize our current achievements as follows. 1) We compared the features of gene transfer using five different AAV serotypes in combination with three different promoters, namely, CMV, mouse CaMKII (CaMKII), and human synapsin 1 (hSyn1), in the marmoset cortex with those in the mouse and macaque cortices. 2) We used target-specific double-infection techniques in combination with TET-ON and TET-OFF using lentiviral retrograde vectors for enhanced visualization of neural connections. 3) We used an AAV-mediated gene transfer method to study the transcriptional control for amplifying fluorescent signals using the TET/TRE system in the primate neocortex. We also established systems for shRNA mediated gene targeting in a neocortical region where a gene is significantly expressed and for expressing the gene using the CMV promoter for an unexpressed neocortical area in the primate cortex using AAV vectors to understand the regulation of downstream genes. Our findings have demonstrated the feasibility of using viral vector mediated gene transfer systems for the study of primate cortical circuits using the marmoset as an animal model. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 354-372, 2017.

Two-photon imaging with genetically encoded calcium indicators (GECIs) enables long-term observation of neuronal activity invivo. However, there are very few studies of GECIs in primates. Here, we report a method for long-term imaging of a GECI, GCaMP6f, expressed from adeno-associated virus vectors in cortical neurons of the adult common marmoset (Callithrix jacchus), a small New World primate. We used a tetracycline-inducible expression system to robustly amplify neuronal GCaMP6f expression and up- and downregulate it for more than 100days. We succeeded in monitoring spontaneous activity not only from hundreds of neurons three-dimensionally distributed in layers 2 and 3 but also from single dendrites and axons inlayer 1. Furthermore, we detected selective activities from somata, dendrites, and axons in the somatosensory cortex responding to specific tactile stimuli. Our results provide a way to investigate the organization and plasticity of cortical microcircuits at subcellular resolution in non-human primates. Long-term two-photon calcium imaging has been challenging in non-human primates. Sadakane etal. use an inducible expression system to visualize cortical neurons in adult marmosets. They show that the same neuronal population can be followed over 100days and neuronal responses to tactile stimulation can be imaged at subcellular resolution.

It is generally thought that orientation selectivity first appears in the primary visual cortex (V1), whereas neurons in the lateral geniculate nucleus (LGN), an input source for V1, are thought to be insensitive to stimulus orientation. Here we show that increasing both the spatial frequency and size of the grating stimuli beyond their respective optimal values strongly enhance the orientation tuning of LGN neurons. The resulting orientation tuning was clearly contrast-invariant. Furthermore, blocking intrathalamic inhibition by iontophoretically administering γ-aminobutyric acid (GABA)A receptor antagonists, such as bicuculline and GABAzine, slightly but significantly weakened the contrast invariance. Our results suggest that orientation tuning in the LGN is caused by an elliptical classical receptive field and orientation-tuned surround suppression, and that its contrast invariance is ensured by local GABAA inhibition. This contrast-invariant orientation tuning in LGN neurons may contribute to the contrast-invariant orientation tuning seen in V1 neurons.

Surround suppression is a phenomenon whereby stimulation of the extraclassical receptive field suppressively modulates the visual responses of neurons in the primary visual cortex (V1) (also known as area 17). It is known that surround suppression tunes to spatial frequencies (SFs) that are much lower and broader than the frequencies to which the classical receptive field tunes. In this study, we tested the effects of varying SFs on surround suppression by using a circular sinusoidal grating patch that covered both the classical receptive field and the extraclassical receptive field. Using area-summation tuning curves, we found high-SF-tuned surround suppression in the cat V1. This high-SF-tuned surround suppression causes the SF tuning to shift to low SF for large stimuli. By simulating a model neuron lacking a suppressive surround mechanism, we confirmed that these preferred SF shifts do not occur in the absence of surround suppression. We surmise that the high-SF-tuned suppression, which shifts the preferred SF according to size, functionally contributes to the scale-invariant processing of visual images in V1.

In the primary visual cortex (V1), the response of a neuron to stimulation of its classical receptive field (CRF) is suppressed by concurrent stimulation of the extraclassical receptive field (ECRF), a phenomenon termed 'surround suppression'. It is also known that the orientation tuning of V1 neurons becomes sharper as the size of the stimulus increases beyond the CRF. However, there have been few quantitative investigations of the relationship between sharpening of orientation tuning and surround suppression. We examined this relationship in 73 V1 neurons recorded from anesthetized and paralysed cats using sinusoidal grating patches as stimuli. We found that sharpening of orientation tuning was significantly correlated with the strength of surround suppression for large stimuli that cover both CRF and ECRF. Furthermore, simulation analysis using a variety of tuning widths and most suppressive orientation of orientation-tuned surround suppression demonstrated that broadly orientation-tuned surround suppression sharpens orientation tuning for large gratings without shift in optimal orientation. Our findings suggest that one of the functional roles of surround suppression in V1 is enhancement of orientation discrimination for large and uniformly patterned objects.

To study the molecular mechanism how cortical areas are specialized in adult primates, we searched for area-specific genes in macaque monkeys and found striking enrichment of serotonin (5-hydroxytryptamine, 5-HT) 1B receptor mRNA, and to a lesser extent, of 5-HT2A receptor mRNA, in the primary visual area (V1). In situ hybridization analyses revealed that both mRNA species were highly concentrated in the geniculorecipient layers IVA and IVC, where they were coexpressed in the same neurons. Monocular inactivation by tetrodotoxin injection resulted in a strong and rapid (

A SOFC system powered by kerosene has been developed. This system consists of desulfurization, reforming section, SOFC and air pre-heater. We designed a thermal self-sustainable SOFC system by using kerosene. The heat balance between SOFC and reforming section can be controlled to change Uf mainly. But we have developed the autothermal reforming catalyst to have some choices for the thermal self-sustainable SOFC system.

Recently, for the purpose of utilizing renewable energy in a community, interactive electricity power networks, such as smart grid, have received much attention. In those electricity power networks, the technologies for maintain the balance of electricity demand and supply in a limited area become more important. Installing photovoltaic system can be effective for supplying electricity in a limited area at the peak load time, but PV output depends on weather conditions. Thus we propose to combine PV power generation with demand response program. We simulate interchange PV power with demand response program in a residential community. Some numerical results, which demonstrate the validity of the proposed method, will be presented.

Two-photon microscopy in combination with a technique involving the artificial expression of fluorescent protein has enabled the direct observation of dendritic spines in living brains. However, the application of this method to primate brains has been hindered by the lack of appropriate labeling techniques for visualizing dendritic spines. Here, we developed an adeno-associated virus vector-based fluorescent protein expression system for visualizing dendritic spinesin vivoin the marmoset neocortex. For the clear visualization of each spine, the expression of reporter fluorescent protein should be both sparse and strong. To fulfill these requirements, we amplified fluorescent signals using the tetracycline transactivator (tTA)–tetracycline-responsive element system and by titrating down the amount of Thy1S promoter-driven tTA for sparse expression. By this method, we were able to visualize dendritic spines in the marmoset cortex by two-photon microscopyin vivoand analyze the turnover of spines in the prefrontal cortex. Our results demonstrated that short spines in the marmoset cortex tend to change more frequently than long spines. The comparison ofin vivosamples with fixed samples showed that we did not detect all existing spines by our method. Although we found glial cell proliferation, the damage of tissues caused by window construction was relatively small, judging from the comparison of spine length between samples with or without window construction. Our new labeling technique for two-photon imaging to visualizein vivodendritic spines of the marmoset neocortex can be applicable to examining circuit reorganization and synaptic plasticity in primates.

In the primary visual cortex (V1), the responses of neurons to stimuli presented in their classical receptive fields (CRFs) are modulated by another stimulus concurrently presented in their surround (receptive field surround, SRF). We studied the nature of the modulatory effects of SRF stimulation with respect to stimulus contrast in cat V1. In 51 V1 neurons studied, large SRF stimuli (40 degreesx30 degrees ) induced only the suppression of responses to CRF stimulation and the suppressive effects became stronger as the contrast for SRF stimulation increased. The contrast sensitivity of SRF suppression did not correlate with that of CRF responses. By independently controlling contrast of CRF and SRF stimuli, we studied whether SRF effects vary with CRF response magnitude. Increasing contrast for CRF stimulation caused an upward shift of the range of effective contrasts for SRF stimulation, indicating that a high contrast for SRF stimulation is required for suppressing strong responses to CRF stimulation at high contrasts. To assess the possible origin of the suppressive SRF effect on V1 neurons, we also investigated the contrast dependency of SRF effects in 28 neurons from the lateral geniculate nucleus. Our results suggest that SRF effects obtained at the subcortical level strongly contribute to those in V1. Taken together, we conclude that along the thalamocortical projections, SRF modulation exhibits a gain-control mechanism that scales the suppressive SRF effect depending on the contrast for CRF stimulation. In addition, SRF effects can be facilitatory at low stimulus contrasts potentially due to the enlargement of the summation field.

In the primary visual cortex (V1), the single-neuron response to a grating stimulus placed in the classical receptive field (CRF) is suppressed by a similar stimulus presented in the CRF surround. To assess the input mechanism underlying the surround suppression, we tested the effects of iontophoretically administered GABAA-receptor antagonist, bicuculline methiodide (BMI), for the 46 V1 neurons in anesthetized cats. First, the stimulus-size tuning curves were studied, with or without BMI administration, for each neuron by changing the size of the grating patch. During the BMI administration, the shape of the normalized size tuning curve did not change considerably. Second, the dependency of surround suppression on the orientation of the surround grating was examined. In the control, the surround suppression showed the clear orientation tuning that peaked at an orientation the same as the optimal orientation of the CRF response. The BMI administration did not change the orientation dependency of surround suppression. We also estimated the relative contribution of excitation and inhibition to the size and orientation tuning of surround suppression. It was concluded that cortical excitation and inhibition were well balanced, having similar tuning profiles for both stimulus size and orientation of the surround grating. Furthermore, surround stimuli used for V1 neurons suppressed the CRF response of neurons in the lateral geniculate nucleus. These results suggest that surround suppression is not primarily attributable to the intracortical inhibition, but because of a reduction of thalamocortical inputs, which drive the cortical excitation and inhibition, and a subsequent decrease in the cortical excitatory interactions.

Distinct anatomical regions of the neocortex subserve different sensory modalities and neuronal integration functions, but mechanisms for these regional specializations remain elusive. Involvement of epigenetic mechanisms for such specialization through the spatiotemporal regulation of gene expression is an intriguing possibility. Here we examined whether epigenetic mechanisms might play a role in the selective gene expression in the association areas (AAs) and the primary visual cortex (V1) in macaque neocortex. By analyzing the two types of area-selective gene promoters that we previously identified, we found a striking difference of DNA methylation between these promoters, i.e., hypermethylation in AA-selective gene promoters and hypomethylation in V1-selective ones. Methylation levels of promoters of each area-selective gene showed no areal difference, but a specific methyl-binding protein (MBD4) was enriched in the AAs, in correspondence with expression patterns of AA-selective genes. MBD4 expression was mainly observed in neurons. MBD4 specifically bound to and activated the AA-selective genes bothin vitroandin vivo. Our results demonstrate that methylation in the promoters and specific methyl-binding proteins play an important role in the area-selective gene expression profiles in the primate neocortex.

We are interested in identifying and characterizing various projection neurons that constitute the neocortical circuit. For this purpose, we developed a novel lentiviral vector that carries the tetracycline transactivator (tTA) and the transgene under the TET Responsive Element promoter (TRE) on a single backbone. By pseudotyping such a vector with modified rabies G-protein, we were able to express palmitoylated-GFP (palGFP) or turboFP635 (RFP) in corticothalamic, corticocortical, and corticopontine neurons of mice. The high-level expression of the transgene achieved by the TET-Off system enabled us to observe characteristic elaboration of neuronal processes for each cell type. At higher magnification, we were able to observe fine structures such as boutons and spines as well. We also injected our retrograde TET-Off vector to the marmoset cortex and proved that it can be used to label the long-distance cortical connectivity of millimeter scale. In conclusion, our novel retrograde tracer provides an attractive option to investigate the morphologies of identified cortical projection neurons of various species.

Animals can make faster behavioral responses to multisensory stimuli than to unisensory stimuli. The superior colliculus (SC), which receives multiple inputs from different sensory modalities, is considered to be involved in the initiation of motor responses. However, the mechanism by which multisensory information facilitates motor responses is not yet understood. Here, we demonstrate that multisensory information modulates competition among SC neurons to elicit faster responses. We conducted multiunit recordings from the SC of rats performing a two-alternative spatial discrimination task using auditory and/or visual stimuli. We found that a large population of SC neurons showed direction-selective activity before the onset of movement in response to the stimuli irrespective of stimulation modality. Trial-by-trial correlation analysis showed that the premovement activity of many SC neurons increased with faster reaction speed for the contraversive movement, whereas the premovement activity of another population of neurons decreased with faster reaction speed for the ipsiversive movement. When visual and auditory stimuli were presented simultaneously, the premovement activity of a population of neurons for the contraversive movement was enhanced, whereas the premovement activity of another population of neurons for the ipsiversive movement was depressed. Unilateral inactivation of SC using muscimol prolonged reaction times of contraversive movements, but it shortened those of ipsiversive movements. These findings suggest that the difference in activity between the SC hemispheres regulates the reaction speed of motor responses, and multisensory information enlarges the activity difference resulting in faster responses., Japan. Ministry of Education, Culture, Sports, Science and Technology (Grant-in-Aid for Scientific Research on Priority Areas (Molecular Brain Science)), Japan. Ministry of Education, Culture, Sports, Science and Technology (Grant-in-Aid for Young Scientists), Japan. Science and Technology Agency (CREST Program)

We previously suggested that orientation-tuned surround suppression of responses of cells in the primary visual cortex (V1) is primarily caused by a decrease in geniculocortical input for the cell [Ozeki H, Sadakane O, Akasaki T, Naito T, Shimegi S, Sato H (2004) Relationship between excitation and inhibition underlying size tuning and contextual response modulation in the cat primary visual cortex. J Neurosci 24:1428-1438]. To further test this hypothesis, we compared the strength of orientation and spatial phase selectivity of surround suppression, and the spatial extent of the extraclassical receptive field (ECRF) between the lateral geniculate nucleus (LGN) and V1 neurons of anesthetized cats. Extraclassical surround suppression in the LGN was well tuned to orientation-contrast and relative spatial phase between the classical receptive field (CRF) and ECRF stimuli. Significant orientation-tuned surround suppression was observed in 72.6% of the LGN neurons and the 66.7% of the V1 neurons tested. The degree of orientation selectivity of ECRF in LGN was comparable to that in V1; however, the strength of the relative spatial phase selectivity of ECRF in LGN was higher than that previously reported for V1 [Akasaki T, Sato H, Yoshimura Y, Ozeki H, Shimegi S (2002) Suppressive effects of receptive field surround on neuronal activity in the cat primary visual cortex. Neurosci Res 43:207-220; DeAngelis GC, Freeman RD, Ohzawa I (1994) Length and width tuning of neurons in the cat's primary visual cortex. J Neurophysiol 71:347-374]. In 70% of the LGN neurons that exhibited significant orientation-tuned extraclassical surround suppression, the effective orientation of the suppression varied according to a change in the orientation of CRF stimulus, while the remaining 30% exhibited a fixed preferred orientation of the suppression regardless of the orientation of the CRF grating. These results suggest that the basic properties of surround suppression, such as orientation and spatial phase tuning, already exist in cat LGN and that a decrease of surround suppression in excitatory inputs from LGN by surround suppression is the primary cause of surround suppression in V1. Corticogeniculate feedback may further elaborate the properties of surround suppression in LGN.

In the primary visual cortex (V1), the responses of neurons to stimuli presented in their classical receptive fields (CRFs) are modulated by another stimulus concurrently presented in their surround (receptive field surround, SRF). We studied the nature of the modulatory effects of SRF stimulation with respect to stimulus contrast in cat V1. In 51 V1 neurons studied, large SRF stimuli (40 degreesx30 degrees ) induced only the suppression of responses to CRF stimulation and the suppressive effects became stronger as the contrast for SRF stimulation increased. The contrast sensitivity of SRF suppression did not correlate with that of CRF responses. By independently controlling contrast of CRF and SRF stimuli, we studied whether SRF effects vary with CRF response magnitude. Increasing contrast for CRF stimulation caused an upward shift of the range of effective contrasts for SRF stimulation, indicating that a high contrast for SRF stimulation is required for suppressing strong responses to CRF stimulation at high contrasts. To assess the possible origin of the suppressive SRF effect on V1 neurons, we also investigated the contrast dependency of SRF effects in 28 neurons from the lateral geniculate nucleus. Our results suggest that SRF effects obtained at the subcortical level strongly contribute to those in V1. Taken together, we conclude that along the thalamocortical projections, SRF modulation exhibits a gain-control mechanism that scales the suppressive SRF effect depending on the contrast for CRF stimulation. In addition, SRF effects can be facilitatory at low stimulus contrasts potentially due to the enlargement of the summation field.

Fuel cell vehicles (FCVs) are an emerging transportation technology, with a potential to provide very low vehicle emissions and significant improvements in fuel efficiency. The choice of a fuel for FCVs must consider several critical issues, including the availability of a distribution and storage infrastructure, manufacturing cost and capital requirements,energy efficiency, and performance. Gasoline, one of the candidate fuels, is noteworthy not only for its existing infrastructure, but because it has the possibility of usage for both FCVs and conventional internal combustion engines. Gasoline consists of different types of hydrocarbons, including paraffins, naphthens, olefins and aromatics - - with carbon numbers distributed over a wide range. Gasoline also contains a variety of sulfur compounds and selected additives in small amounts., In some cases gasoline also contains oxygenates such as MTBE and ethanol. To develop the optimal fuels and associated catalysts for FCVs it is necessary to understand the effects of these constituents on FCV fuel processing. Reforming performance has been evaluated with a variety of gasoline components, as well as conventional gasoline provided by refineries. Tests of initial reformer catalyst activity and durability at auto-thermal reforming (ATR) conditions have been conducted. Results showed similar reforming performance among most hydrocarbon components except aromatics. The addition of MTBE improved reforming reactivity. Both aromatics and sulfur compounds caused rapid deterioration of reforming activity, while the magnitude of the reactivity loss varied between different aromatic compounds.

Neurons of the primary visual cortex (V1) respond to stimuli presented at the classical receptive field (CRF), and the responses are suppressively modulated by stimuli simultaneously presented at the receptive field surround (SRF) [1, 2]. The response modulation by an SRF stimulus depends on the stimulus orientation and spatial frequency; thus, it was suggested that intracortical mechanisms contribute to this phenomenon [2]. We recently reported that this suppressive modulation is not due to a direct inhibition to the cell by the intracortical inhibition [3] (also see the chapter by H. Ozeki et al., this volume).

To assess the network dynamics in the primary visual cortex, we performed simultaneous recordings of neuronal activity from cell pairs within 1 mm apart in anesthetized and paralyzed cats. Cross-correlograms (CCs) were calculated for the responses to grating stimuli with varying orientations and sizes for the 30 cell pairs to date. There were three patterns of cell pairs in respect of tuning property to stimulus sizes. First, both cells exhibited strong suppression of firings with stimuli larger than their classical receptive field (CRF). Second, only one cell exhibited suppression to the large stimuli while the other did not. In these two patterns, the contribution of the peak CCs became smaller when the grating stimulus was larger than the CRFs. And third, both cells did not significantly reduce activity to the grating patch larger than their CRFs. In this case, the peak contribution in CCs did not vary with an increase of the stimulus size. We concluded that the strength of firing interaction is positively correlated with the change in firing rate of cell pairs because of the change of stimulus sizes. We also analyzed CCs in respect of orientation preference of cell pairs. Although the significant peak CCs were most often observed for the common and optimal orientation of the cell pairs, there were some pairs that showed strong peaks for the nonpreferred orientation (more than 60° apart from the optimal) of stimulus, which evoked responses with low firing levels. This result suggests that the strength of firing correlation of cell pairs does not simply depend on the firing rate when the stimulus orientation is varied.

The response of neurons in the primary visual cortex (V1) is modulated by the stimulus presented at the surround of the classical receptive field (CRF), and the predominant effect of this surround stimulation is suppressive [1–5]. The strength of the surround suppression is dependent on the relationship of stimulus parameters, such as orientation and spatial frequency, between grating stimuli at the inside and outside of the CRF [1, 2]. Although properties of the surround suppression have been well characterized, input mechanisms underlying the stimulus-context-dependent response modulation and the size-tuning properties of V1 neurons have not been clarified yet. There is evidence that the surround suppression in VI is both sub-cortical [5] and intracortical [2, 4] in origin.