Hiroyuki Ichijo

Regional expression of a gene encoding a neuron-specific Na(+)-dependent inorganic phosphate cotransporter (DNPI) in the rat forebrain.

We have analyzed expression of a gene encoding a brain-specific Na(+)-dependent inorganic phosphate cotransporter (DNPI), which was recently cloned from human brain, in rat forebrain using in situ hybridization. The expression of DNPI mRNA showed a widespread but highly heterogeneous pattern of distribution in the forebrain, where hybridization signals were observed in neurons but not in any other types of cells. Neurons expressing the mRNA were far more numerous in the diencephalon than in the telencephalon. In the thalamus, a number of neurons with high levels of signals were localized to all nuclei of the dorsal thalamus, habenular nuclei and subthalamic nucleus, but not the reticular nucleus and zona incerta. Moderate signal levels were seen in many neurons throughout the hypothalamus, particularly the ventromedial, paraventricular, supraoptic and arcuate nuclei, lateral hypothalamic area and mammillary complex. In contrast, expression of DNPI mRNA in the telencephalon was generally at a low level and occurred locally in some restricted regions within the neocortex, retrosplenial cortex, piriform cortex, olfactory regions, hippocampal formation and medial amygdaloid nucleus. The present results suggest that DNPI functions in heterogeneous neuron populations as a neuron-specific Na(+)-dependent inorganic phosphate cotransport system predominantly expressed in the diencephalon of the rat.

Proteoglycans as cues for axonal guidance in formation of retinotectal or retinocollicular projections.

Understanding the formation of neuronal circuits has long been one of the basic problems in developmental neurobiology. Projections from the retina to their higher center, the optic tectum in nonmammalian vertebrates and the superior colliculus in mammals, are most amenable to experimental approaches; thus, much information has been accumulated about the mechanisms of axonal guidance. The retinal axons navigate along the appropriate pathway with the help of a series of guidance cues. Although much of the work has focused on proteinaceous factors, proteoglycans have been identified as playing important roles in retinal axon guidance. Chondroitin sulfate proteoglycans and heparan sulfate proteoglycans are involved in essential decisions of axon steering along the pathway. However, it has not been determined whether diversity of the carbohydrate chains results in differential effects and how their diversity is recognized by growth cones, which represent an important area of future research.

Differentiation of the chick retinotectal topographic map by remodeling in specificity and refinement in accuracy

To understand the development of the retinotopic map, differentiation of the topographic map was quantitatively examined in the chick. Labeling the retinal ganglion cell (RGC) axons anterogradely with the local injections of DiI revealed the relative anteroposterior positions of their growth cones (GCs) on the tecta as a function of the nasotemporal positions of the injected sites in the retinae, which allowed a graphic representation of the map. The topographic map was depicted by combination of two parameters: specificity which indicates strictness of the topographic relationship between locations of the RGC bodies and their GCs on the tectum, and accuracy which indicates an extent of the GC displacement on the tectum. A crude projection with low specificity emerged at embryonic day 11 (E11). The initial crude projection was remodeled into the inaccurate map with high specificity by E13; thereafter, it was refined to the accurate map with higher specificity by E15. The results suggest that the elements of the guidance mechanism operate stage by stage through the formation of the crude projection, the remodeling in specificity, and the refinement in accuracy to establish the final topographic map.

Lateralization, maturation, and anteroposterior topography in the lateral habenula revealed by ZIF268/EGR1 immunoreactivity and labeling history of neuronal activity

We report habenular lateralization in a simple transgenic mouse model used for labeling a facet of neuronal activity history. A transgenic construct comprised of a zif268/egr1 immediate-early gene promoter and a gene for normal Venus fluorescent protein with a membrane tag converted promoter activity into long-life fluorescent proteins, which was thought to describe a facet of neuronal activity history by summing neuronal activity. In addition to mapping the immediate-early gene-immunopositive cells, this method helped demonstrate the functionality of the lateral habenular nucleus (LHb). During postnatal development, the LHb was activated between postnatal days 10 and 16. The water-immersion restraint stress also activated the LHb over a similar period. LHb activation was functionally lateralized, but had no directional bias at the population level. Moreover, the posterior LHb was activated in the early stage after the stress, while the anterior LHb was activated in the later stage. Our results indicate lateralization, maturation, and anteroposterior topography of the LHb during postnatal development and the stress response.

Moderate repulsive effects of E-unit-containing chondroitin sulfate (CSE) on behavior of retinal growth cones

Chondroitin sulfate (CS), the carbohydrate chain of chondroitin sulfate proteoglycans, is involved in neuronal circuit formation during development. CS shows great structural diversity with combination of disaccharide units of different structure (A-, C-, D-, or E-unit). However, whether its structural diversity contributes to pathway formation remains unclear. We chemically coupled the reducing end of various types of CS to the amino group of phosphatidylethanolamine (lipid-derivatized CS, CS-PE) and established an in vitro time-lapse assay to observe the behaviors of growth cones of retinal ganglion cells from embryonic day 6 chick retina on exposure to beads coated with lipid-derivatized CS (CS-PE beads). Among CS-PEs with different content of the structural units, the beads coated with E-unit-containing CS-PE [E-unit: GlcAβ1-3GalNAc(4,6-O-disulfate)] (CSE-PE beads) significantly caused the growth cones to retract and to turn away from the beads, but the beads coated with CSA-, CSC- or CSD-PE beads did not. Importantly, not all the growth cones retracted equally from the CSE-PE beads, but they showed continuum of the repulsive behaviors; some behaved moderately and others remarkably. The growth cones distinguished different samples of CS: CSE and the others. Moreover, the continuum of the repulsive behaviors suggests that CS might be involved with the fine regulation of growth cones behavior through its characteristic structure.

Restricted distribution of D-unit-rich chondroitin sulfate carbohydrate chains in the neuropil encircling the optic tract and on a subset of retinal axons in chick embryos

To obtain basic information about the structural diversity and functional specificity of chondroitin sulfates (CSs) in the formation of the retinotectal pathway in chick embryos, the distribution of CSs around the optic tract was investigated by using anti‐CS monoclonal antibodies with different specificities. The CSs are unbranched polymers composed of repeating disaccharide units of glucuronic acid (GlcA) and N‐acetylgalactosamine (GalNAc). The disaccharide units are classified into O‐, A‐, C‐, D‐, and E‐units based on the position(s) of the added sulfate group(s). The MO‐225 monoclonal antibody recognizes CSs that are rich in the D‐unit [GlcA(2S)β1–3GalNAc(6S)]; the MO‐225 epitopes were distributed in the diencephalotelencephalic boundary and the neuropil encircling the optic tract. In addition, they were distributed on membrane surfaces of the retinal axons running in an interface layer in contact with the neuropil encircling the optic tract. The results suggest that D‐unit‐rich CSs are involved in delimiting the border of the optic tract and in the chronological sorting of the retinal axons. J. Comp. Neurol. 495:470–479, 2006.

An Evolutionary Hypothesis of Binary Opposition in Functional Incompatibility about Habenular Asymmetry in Vertebrates

Many vertebrates have asymmetrical circuits in the nervous system. There are two types of circuit asymmetry. Asymmetrical circuits in sensory and/or motor systems are usually related to lateralized behaviors. It has been hypothesized that spatial asymmetry in the environment and/or social interactions has led to the evolution of asymmetrical circuits by natural selection. There are also asymmetrical circuits that are not related to lateralized behaviors. These circuits lie outside of the sensory and motor systems. A typical example is found in the habenula (Hb), which has long been known to be asymmetrical in many vertebrates, but has no remarkable relationship to lateralized behaviors. Instead, the Hb is a hub wherein information conveyed to the unilateral Hb is relayed to diverging bilateral nuclei, which is unlikely to lead to lateralized behavior. Until now, there has been no hypothesis regarding the evolution of Hb asymmetry. Here, we propose a new hypothesis that binary opposition in functional incompatibility applies selection pressure on the habenular circuit and leads to asymmetry. Segregation of the incompatible functions on either side of the habenula is likely to enhance information processing ability via creating shorter circuits and reducing the cost of circuit duplication, resulting in benefits for survival. In zebrafish and mice, different evolutionary strategies are thought to be involved in Hb asymmetry. In zebrafish, which use a strategy of structurally fixed asymmetry, the asymmetrical dorsal Hb leads to constant behavioral choices in binary opposition. In contrast, in mice, which use a strategy of functionally flexible lateralization, the symmetrical lateral Hb is functionally lateralized. This makes it possible to process complicated information and to come to variable behavioral choices, depending on the specific situation. These strategies are thought to be selected for and preserved by evolution under selection pressures of rigidity and flexibility of sociability in zebrafish and mice, respectively, as they are beneficial for survival. This hypothesis is highly valuable because it explains how the Hb evolved differently in terms of asymmetry and lateralization among different species. In addition, one can propose possible experiments for the verification of this hypothesis in future research.

Axons from the medial habenular nucleus are topographically sorted in the fasciculus retroflexus.

Abstract We generated transgenic mice lines with a construct consisting of the zif268/egr1 promoter and the gene for the normal long-life yellow fluorescent protein (Venus) with a membrane localization sequence. One of the lines exhibited topographic labeling in the medial habenular nucleus (MHb) during postnatal development, which confirmed the previous findings that the medial, lateral, and dorsal areas of MHb project to the ventral, dorsal, and lateral parts of the interpeduncular nucleus, respectively. In addition, the membranous localization of the labeling allowed us to observe spacial arrangement of the labeled axons in the fasciculus retroflexus (FR) in the transgenic mice. Here, we report topographic sorting of the MHb axons in the FR. At postnatal day (P) 5 and P10, the labeled axons from the medial MHb were fasciculated and ran through the narrow path in the core of the FR. At P24, the labeled axons from the medial and dorsal MHb were fasciculated and ran through the broad path in the FR core. No labeling occurred in the lateral MHb throughout development; correspondingly, parts of the FR core remained unlabeled. The results indicated that the axons from the medial and dorsal areas of the MHb are grouped together in the FR of this transgenic line and are sorted out from the axons from the lateral MHb.

Influences of retinal axons on the cultural substrate containing biotin-conjugated chondroitin sulfate in vitro

Although chondroitin sulfate (CS) is known to act as an inhibitory axon guidance cue, retinal axons show substantial growth on a culture substrate containing CS. Thus, the question arises as to how retinal axons elongate on CS-containing culture substrates. To elucidate the effects of retinal axons on a substrate containing CS, we synthesized biotinylated CS (biotin-CS) and developed a culture substrate with streptavidin-conjugated biotin-CS (complex between streptavidin and biotin-CS) to culture retinal axons. The effects of retinal axons on the streptavidin–biotin-CS complex were analyzed immunocytochemically using antibodies against CS and streptavidin, which recognize the carbohydrate and protein portions of the complex, respectively. After the axons were cultured on the substrate, areas that were CS-immunonegative but streptavidin-immunopositive were observed on the surface, corresponding to areas with or without axons, respectively. Absence of CS immunostaining was considered to be caused by structural alterations in the carbohydrate chains of the CS under the influence of the axons.

Labeling history of neuronal activity and functional asymmetry of fasciculus retroflexus

s / Neuroscience Research 58S (2007) S1–S244 S69 O2P-KØ3 Development of dendritic membrane-targeting signals using lentiviral vectors Hiroshi Kameda1, Hiroyuki Hioki1, Takahiro Furuta1, Koji Ohira1, Wakoto Matsuda1, Kouichi Nakamura1,2, Takeshi Kaneko1,2 1 Department of Morphology, Brain Science, Kyoto University, Kyoto, Japan; 2 CREST, JST, Japan We previously reported that lentivirus with synapsin I promoter expresses green fluorescent protein (GFP) specifically in neurons. The expression was, however, so weak that neuronal processes were not well visualized. In the present study, we tried to develop dendritic membrane-targeted GFP to label the infected neurons in a Golgi stain-like manner. Fatty acylation sites were first tested for membrane-targeting of GFP. GFP with Fyn N-terminal myristoylation/palmitoylation site (myrGFP) was localized to the dendritic membrane more efficiently than GFP with GAP43 N-terminal palmitoylation site. However, myrGFP was distributed at not only dendritic, but also axonal membranes. Thus, we next examined six kinds of putative dendrite-targeting signals. Of these signals, C-terminal of low density lipoprotein receptor (LDLRCT) was the most efficient in targeting myrGFP to the dendritic membrane. Furthermore, we generated transgenic mice expressing myrGFP-LDLRCT and investigated the effect of the dendritic membrane-targeting signals. O2P-KØ4 Labeling history of neuronal activity and functional asymmetry of fasciculus retroflexus Hiroyuki Ichijo1, Takeharu Nagai2, Michito Hamada1, Makoto Kobayashi3, Satoru Takahashi1 1 Department of Anat Embryology, University of Tsukuba, Tsukuba, Japan; 2 Hokkaido University, Sapporo, Japan; 3 TARA Center, University of Tsukuba, Japan Experiences are encoded in neuronal circuits as their neuronal activity; however, to elucidate structure and function of the responsible circuits has been difficult. Immediate early genes (IEGs) promoters are useful for converting neuronal activity into gene expression. To label experiences, we designed genetic tracers with a fluorescent protein on cellular membrane (e.g. GAP43-Venus) under the control of the IEGs promoters (e.g. zif268 promoter) and generated their transgenic mice. Because the GAP43-Venus has long life-time, it is accumulated on neurons activated; thus, the gene constructs operate to sum the neuronal activity and to label the experiences. The labeled sites and their fluorescent intensity represent quality and quantity of the experiences, respectively. Here we report that fasciculus retroflexus sinistra was transiently labeled asymmetrically on postnatal day 13 afterward in the transgenic mice, which indicates validity of the strategy and functional asymmetry of CNS. Research funds: KAKENHI 13210021 O2P-KØ5 Isolation of song nuclei specific genes by using cDNA microarray in Bengalese finch Masaki Kato, Kazuo Okanoya Laboratory for Biolinguistics, BSI, RIKEN, Saitama, Japan Songbirds, like humans, are unusual animals in that they exhibit vocal learning and song syntax complexity. We studied the molecular mechanisms of song syntax complexity in the Bengalese finch using gene expression profiles with cDNA microarray. We constructed a Bengalese finch cDNA microarray containing 3840 independent known genes. We tried to isolate the song nuclei specific genes using the cDNA microarray, as the song nuclei are neural substrate for birdsong. The HVC is an especially important nucleus in vocal learning and singing behavior. Therefore, we focused on isolating HVC-specific genes. The HVC and caudal nidopallium (cNidopallium: neighboring areas of the HVC) in adult male Bengalese finches were microdissected and the gene expression patterns were compared using two-color competitive microarray hybridization. We identified 34 genes highly expressed in the HVC and some genes were confirmed by in situ hybridization. Consequently, we found 15 genes that were expressed strongly in the HVC compared to the cNidopallium, and most of these genes were also expressed in other song nuclei. O2P-KØ6 Temporal relations of auditory selectivity among song-related thalamo-cortical brain loop nuclei in Bengalese finches Yoshimasa Seki1,2, Kazuo Okanoya2 1 Graduate School of Science and Technology, Chiba University, Chiba, Japan; 2 Lab. for Biolinguistics, RIKEN-BSI, Wako, Japan There are a set of brain nuclei in songbirds that are related with song learning and production. Among these, HVC and mMAN represents a part of the thalamo-cortical loop connection, and output of HVC is fed to the input of mMAN via thalamus, output of mMAN then becomes input to HVC. Here we report first electrophysiological study of the auditory response of mMAN neurons and correlations with the activities of HVC neurons in anesthetized Bengalese finch males. The auditory responses of mMAN neurons showed strong BOS (bird’s own song) selectivity that rivals to HVC. Event correlation data from simultaneous recordings of mMAN and HVC neurons showed that some activities of mMAN neurons preceded the firing of HVC neurons. These findings are in parallel with anatomical connection between the two nuclei and suggest that mMAN neurons could modulate some activities of HVC. Research funds: Work partially supported by the Sasakawa Scientific Research Grant from The Japan Science Society O2P-KØ7 Spatial temporal credit assignment neural network model Adam P.D. Ponzi DEI Lab, RIKEN BSI, Saitama, Japan In Ponzi (2006), I presented a model where replayed recurrent activity in an association layer generated at goals provided a novel way to form “context” cells representing a value function on experienced paths within the given action selection policy. Indeed replay of experienced place cells in CA3 has been observed by Foster and Wilson. The model is based on the loop projections from the hippocampus and cortex (pFC) to the striatum with feedback from SNr/VTA and uses a novel threshold LTP/LTD model of the D1/D2 dopamine receptors, compatible with Goto and Grace results of a balance of tonic and phasic dopamine at the hippocampal and pFC accumbens synapses. Here I show the modifiable action selection system (Ponzi, 2007a) taught by the context cell learns the correct policy and makes a transition from exploratory to goal directed behaviour (Ponzi, 2007b) in both egocentric and allocentric plus maze tasks with multiple variable reward magnitude goals. The observed response of task and expert neurons of the dorsal and ventral striatum, and some behavioural characteristics, are reproduced. Refs. Ponzi, A., IEICE NC Technical Report. 103;163 19-24 (July 2006); Ponzi, A., IJCNN 2007a; Ponzi, A., CNS 2007b abstract. O2P-KØ8 Can theta burst transcranial magnetic stimulation affect cortico-muscular synchronization in humans? Murat Saglam1, Hayato Yanagida1, Kaoru Matsunaga2, Nobuki Murayama1, Yuki Hayashida1, Ryoji Nakanishi2 1 Department of Graduate School of Science and Technology, Kumamoto University, Japan; 2 Department of Neurology, Kumamoto Kinoh Hospital, Japan Recently it has shown that a novel paradigm of repetitive transcranial magnetic stimulation (rTMS), ‘theta-burst stimulation’ (TBS) can induce long-lasting suppression/facilitation of the neuronal excitability in humans, even with the stimulation duration shorter than those of conventional rTMS protocols. We examined effects of TBS on the functional coupling between left motor cortex and right first dorsal interosseous (FDI) muscle by means of the coherence between electroencephalography (EEG) and electromyography (EMG). Before and after the delivery of TBS over either primary motor area (PMA) or sensory motor area (SMA), EMG from FDI muscle and EEG from 20 scalp sites were recorded while the subject performed a weak isometric hand contraction. EEG-EMG coherence localizing for the C3 scalp site and at beta band (13–30 Hz) significantly suppressed 30–60 min after the TBS on PMA, but not that on SMA, and recovered to the original level in 90–120 min.