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During sleep and awake rest, the neocortex generates large-scale slow-wave (SW) activity. Here, we report that the claustrum coordinates neocortical SW generation. We established a transgenic mouse line that enabled the genetic interrogation of a subpopulation of claustral glutamatergic neurons. These neurons received inputs from and sent outputs to widespread neocortical areas. The claustral neuronal firings mostly correlated with cortical SW activity. In vitro optogenetic stimulation of the claustrum induced excitatory postsynaptic responses in most neocortical neurons, but elicited action potentials primarily in inhibitory interneurons. In vivo optogenetic stimulation induced a synchronized down-state featuring prolonged silencing of neural activity in all layers of many cortical areas, followed by a down-to-up state transition. In contrast, genetic ablation of claustral neurons attenuated SW activity in the frontal cortex. These results demonstrate a crucial role of claustral neurons in synchronizing inhibitory interneurons across wide cortical areas for the spatiotemporal coordination of SW activity.

Escape responses to threatening stimuli are vital for survival in all animal species. Larval zebrafish display fast escape responses when exposed to tactile, acoustic, and visual stimuli. However, their behavioral responses to chemosensory stimuli remain unknown. In this study, we found that carbon dioxide (CO2) induced a slow avoidance response, which was distinct from the touch-evoked fast escape response. We identified the gonadotropin-releasing hormone 3-expressing terminal nerve as the CO2sensor in the nose. Wide-field calcium imaging revealed downstream CO2-activated ensembles of neurons along three distinct neural pathways, olfactory, trigeminal, and habenulo-interpeduncular, further reaching the reticulospinal neurons in the hindbrain. Ablation of the nose, terminal nerve, or trigeminal ganglion resulted in a dramatic decrease in CO2-evoked avoidance responses. These findings demonstrate that the terminal nerve-trigeminal system plays a pivotal role in triggering a slow chemosensory avoidance behavior in the larval zebrafish.

Spatiotemporal control of gene expression is important for neural development and function. Here, we show that heterogeneous nuclear ribonucleoprotein (hnRNP) A/B is highly expressed in developing olfactory sensory neurons (OSNs), and its knockout results in reduction in mature OSNs and aberrant targeting of OSN axons to the olfactory bulb. RNA immunoprecipitation analysis reveals that hnRNP A/B binds to a group of mRNAs that are highly related to axon projections and synapse assembly. Approximately 11% of the identified hnRNP A/B targets, including Pcdhaand Ncam2, encode cell adhesion molecules. In Hnrnpabknockout mice, PCDHA and NCAM2 levels are significantly reduced at the axon terminals of OSNs. Furthermore, deletion of the hnRNP A/B-recognition motif in the 3′ UTR of Pcdhaleads to impaired PCDHA expression at the OSN axon terminals. Therefore, we propose that hnRNP A/B facilitates OSN maturation and axon projection by regulating the local expression of its target genes at axon terminals.

Pheromones play vital roles for survival and reproduction in various organisms. In many fishes, prostaglandin F2αacts not only as a female reproductive hormone, facilitating ovulation and spawning, but also as a sex pheromone inducing male reproductive behaviors. Here, we unravel the molecular and neural circuit mechanisms underlying the pheromonal action of prostaglandin F2αin zebrafish. Prostaglandin F2αspecifically activates two olfactory receptors with different sensitivities and expression in distinct populations of ciliated olfactory sensory neurons. Pheromone information is then transmitted to two ventromedial glomeruli in the olfactory bulb and further to four regions in higher olfactory centers. Mutant male zebrafish deficient in the high-affinity receptor exhibit loss of attractive response to prostaglandin F2αand impairment of courtship behaviors toward female fish. These findings demonstrate the functional significance and activation of selective neural circuitry for the sex pheromone prostaglandin F2αand its cognate olfactory receptor in fish reproductive behavior.

Endocrine-disrupting chemicals are prevalent in the environment and can impair reproductive success by affecting the hypothalamic-pituitary-gonadal axis. The developing pituitary gland is sensitive to exposure to endocrine-disrupting chemicals, such as bisphenol A (BPA), and sex-specific effects can occur. However, effects on the critical window of neonatal pituitary gland development in mice have not been explored. Therefore, this study determined baseline gene expression in male and female pituitaries and consequences of environmental exposure to 17β-estradiol (E2) and BPA on transcription of genes exhibiting sex differences during the neonatal period. Through microarray and quantitative RT-PCR analysis of pituitaries at postnatal day (PND)1, 3 genes were differentially expressed between males and females: Lhb, Fshb, and intracellular adhesion molecule-5 (Icam5). To see whether E2 and BPA exposure regulates these genes, pituitaries were cultured at PND1 with 10−8M E2 or 4.4 × 10−6M BPA. E2 decreased expression of Lhb, Fshb, and Icam5mRNA in females but only significantly decreased expression of Icam5in males. BPA decreased expression of Icam5similarly to E2, but it did not affect Lhbor Fshb. Importantly, in vivo exposure to 50-μg/kg · d E2 from PND0 to PND7 decreased expression of Lhb, Fshb, and Icam5mRNA in both males and females, whereas 50-mg/kg · d BPA exposure during the same time frame decreased expression of Icam5in females only. Overall, we have uncovered that genes differentially expressed between the sexes can be regulated in part by hormonal and chemical signals in vivo and directly at the pituitary and can be regulated in a sex-specific manner.

Chronic exposure to sphingosine‐1‐phosphate within, or outside, mast cells causes gene expression alterations that modulate FcɛRI‐mediated mast cell responses, and the onset of allergic reaction. Both genes and the environment are determinants in the susceptibility to allergies and may alter the severity of the disease. We explored whether an increase in the levels of the lipid mediator S1P in vivo, a condition found during allergic asthma, could affect the sensitivity or the response of MCs to IgE/Ag and the onset of allergic disease. We found that increasing S1P levels by genetic deletion of S1P lyase, the enzyme catabolizing S1P, led to elevated activity of circulating tryptase. Accordingly, MCs of S1P lyase‐deficient mice were mostly degranulated in the tissues and showed enhanced calcium levels, degranulation, and cytokine production in response to IgE/Ag in vitro. Th 1‐skewed mice (C57BL/6) had lower levels of S1P in circulation and histamine responses than did Th 2‐skewed (129/Sv) mice. However, when S1P levels were increased by pharmacologic inhibition of S1P lyase, the C57BL/6 mice showed increased histamine release into the circulation and anaphylactic responses similar to those in the 129/Sv mice. Culturing of MCs in the presence of S1P enhanced their degranulation responses, and when the S1P‐treated MCs were used to reconstitute MC‐deficient (KitW‐sh) mice, they caused enhanced anaphylaxis. Gene expression arrays in S1P lyase‐deficient MCs and MCs treated with S1P continuously revealed increased expression of numerous genes, including the adhesion molecule CNTN4,which contributed to the enhanced responses. Our findings argue that dysregulation in the metabolism of S1P is a contributing factor in modulating MC responsiveness and the allergic response.

NCAM in vertebrates and its related molecules, apCAM in Aplysia,fasciclin II in Drosophila,and OCAM in mammals, play key roles in various aspects of brain development and functions. In this study, we have identified and characterized three members of the NCAM gene family in zebrafish, designated as zNCAM, zOCAM, and zPCAM. Three molecules exhibit similar domain organization: an amino-terminal signal peptide, five immunoglobulin-like domains, two fibronectin type III-like domains, a transmembrane segment, and a carboxy-terminal cytoplasmic region. A novel molecule zPCAM is most closely related to zNCAM with 66% amino acid identity. Diversity in the extracellular region of zPCAM is generated by insertion of two different types of variable alternatively spliced exons. In situhybridization analysis revealed that three molecules were specifically expressed by the central and peripheral nervous systems from early developmental stages in region-specific and cell-type-specific manners. For example, zPCAM showed a neuromere-specific segmental expression pattern, while zOCAM first appeared in specific clusters of secondary neurons in the forebrain. These results suggest that each member of the NCAM gene family plays distinct roles in the formation and maintenance of functional neuronal networks in the zebrafish nervous system.

Zonal organization is one of the characteristic features observed in both main and accessory olfactory systems. In the main olfactory system, most of the odorant receptors are classified into four groups according to their zonal expression patterns in the olfactory epithelium. Each group of odorant receptors is expressed by sensory neurons distributed within one of four circumscribed zones. Olfactory sensory neurons in a given zone of the epithelium project their axons to the glomeruli in a corresponding zone of the main olfactory bulb. Glomeruli in the same zone tend to represent similar odorant receptors having similar tuning specificity to odorants. Vomeronasal receptors (or pheromone receptors) are classified into two groups in the accessory olfactory system. Each group of receptors is expressed by vomeronasal sensory neurons in either the apical or basal zone of the vomeronasal epithelium. Sensory neurons in the apical zone project their axons to the rostral zone of the accessory olfactory bulb and form synaptic connections with mitral–tufted cells belonging to the rostral zone. Signals originated from basal zone sensory neurons are sent to mitral–tufted cells in the caudal zone of the accessory olfactory bulb. We discuss functional implications of the zonal organization in both main and accessory olfactory systems.

The glomerular sheet in the olfactory bulb (OB) provides an olfactory sensory map identifying which odorant receptors (ORs) in the nose are activated by inhaled odorants. How are the glomeruli spatially arranged in the OB? Using OCAM and neuropilin-1 (NP1) as molecular markers for target glomeruli of distinct subsets of olfactory axons, we demonstrate here that glomeruli are parceled into topographically distinct domains. Spatial arrangement of these domains suggests that each OB contains two mirror-image maps of the glomeruli. In situhybridization shows that the glomeruli representing the same OR are symmetrically arranged; one in a domain in the lateral hemisphere and the other in a corresponding domain in the medial hemisphere of the OB. These results suggest that OB contains two symmetrical OR maps with similar domain organization.

Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a member of the immunoglobulin superfamily expressed on telencephalic neurons, and serves as a ligand for the leukocyte integrin CD11a/CD18. We studied here the binding site in ICAM-5 for CD11a/CD18. Protein constructs containing the first immunoglobulin domain of ICAM-5 were able to support CD11a/CD18 interaction, while deletion of the first domain abolished binding. Monoclonal antibodies reacting with the first domain of ICAM-5 also completely blocked the interaction. The soluble first domain of ICAM-5 inhibited the binding of T cells to immobilized ICAM-5 at concentrations of 50 nM and higher. Interestingly, the sixth domain of ICAM-5 was also able to support leukocyte binding, but this binding activity may not involve leukocyte integrins. To test the involvement of ICAM-5 in leukocyte-neuron interactions, an assay using human T cells binding to rat hippocampal neurons was established. This binding was blocked by monoclonal antibodies against CD11a/CD18 and ICAM-5. Thus ICAM-5 may act as a major adhesion molecule for leukocyte binding to neurons in the central nervous system.

We have isolated a mouse cDNA which shows significant sequence similarity to the yeast nifS‐like gene (y‐NFS1), and termed it m‐Nfs1. The deduced protein sequence (459 amino acids long) has several characteristic features common to those of bacterial NifS proteins, but distinct from them by its amino‐terminal extension which contains a typical mitochondrial targeting presequence. m‐Nfs1 was found to be a soluble 47‐kDa protein in the matrix fraction of mouse liver mitochondria. The m‐Nfs1gene was ubiquitously expressed in most tissues, suggesting its housekeeping function in vivo. We also found that the y‐NFS1 protein was localized in the mitochondrial matrix in yeast cells. These results suggest that both eukaryotic NifS‐like proteins may play some roles in mitochondrial functions.

TWO functional subsets of vomeronasal sensory neurons project their axons to two segregated zones in the accessory olfactory bulb (AOB). Using immunohistochemical methods with antibodies against the novel cell adhesion molecule OCAM, we provide evidence that the segregation of functional pathways is maintained at the level of mitral/tufted (M/T) cells of the mouse AOB and that this pattern emerges early in ontogeny. During embryonic and postnatal development OCAM was strongly expressed by M/T cells in the caudal zone of the AOB where OCAM-negative vomeronasal axons terminated. In contrast, rostral zone M/T cells innervated by OCAM-positive vomeronasal axons displayed no or faint OCAM immunoreactivity. Differential expression of OCAM in segregated M/T cell pathways suggests that OCAM may be involved in defining compartments of connectivity and setting up functional subdivisions in the developing AOB.

Confocal laser scanning imaging was used to reconstruct the three-dimensional distribution of the aggregates of olfactory receptor axons terminating within individual glomeruli in the rabbit main olfactory bulb. Two monoclonal antibodies, R2D5 and R4B12, were used to mark selectively the olfactory receptor axons. Thick coronal sections (100-200 µm. in thickness) through the bulb were labeled with either of the antibodies, and then serial optical sectioning was performed to reconstruct three-dimensional optic images of the labeled axons in the glomeruli. The results revealed an intricate internal structure of the glomeruli with a mesh-like arrangement of bundles of terminal olfactory axons. Developmental study showed that primitive glomeruli lacking the internal structure of the adult form first appear in the 22-day embryo and that the characteristic internal structure of the glomeruli develops mainly during postnatal days. The confocal laser scanning image method together with specific molecular markers provides a simple tool for the three-dimensional analysis of the glomerular structure. Copyright 1994, 1999 Academic Press

TELENCEPHALIN (TLCN) is a cell adhesion molecule belonging to the immunoglobulin superfamily whose expression is restricted to neurons within the most highly developed brain segment, telencephalon. Immunoelectronmicroscopic study revealed that in the hippocampal CA1 region, TLCN was localized at the surface membrane of postsynaptic spines of pyramidal cell dendrites but not at that of axonal terminals. Blocking of TLCN function using anti-TLCN antibody or recombinant soluble TLCN protein caused a striking suppression of the long-term potentiation (LTP) at the Schaffer collateral–CA1 synapses. The suppression was observed even when the blocking was initiated immediately after the tetanic stimuli. These observations suggest a role for TLCN-mediated cell—cell interactions as a key step in the development of LTP.

Telencephalin is a cell adhesion molecule belonging to the immunoglobulin (Ig) superfamily, whose expression is restricted to subsets of neurons in the telencephalon, the most rostral segment of brain. Of all the Ig superfamily molecules so far identified, the structure of telencephalin is most closely related to those of intercellular adhesion molecules (ICAMs)-1 and -3. Here we report the cloning, characterization, and chromosomal localization of the mouse telencephalin gene (Tlcn). TheTlcngene spanned about 6.3 kb and consisted of 11 exons. A signal peptide and individual nine Ig-like domains of telencephalin were encoded by a single exon, while the transmembrane and cytoplasmic regions were fused in a same exon. The primer extension technique was used to establish that the transcription initiation sites were located 92–95 bp upstream from the ATG start codon. DNA sequencing of the 5′-flanking region revealed the presence of a strong initiator element for TATA-less genes, two CAAT boxes, and numerous potential transcription factor binding sites including four E-box and two N-box sequences. Interspecific backcross analysis demonstrated that theTlcngene was mapped in the proximal region of mouse chromosome 9 in close vicinity to theIcam-1gene, suggesting thatTlcnandIcam-1are derived from a common ancestral gene by gene duplication.

A novel method of dipeptide synthesis is described that can be carried out in aqueous solution and does not require complicated protecting and deprotecting procedures. An analgesic neuropeptide named kyotorphin, H-Tyr-Arg-OH, was synthesized from unprotected tyrosine and arginine in a new enzymatic reaction catalyzed by immobilized tyrosyl-tRNA synthetase from Bacillus stearothermophilus. The reaction could be a useful tool in the syntheses of radioisotope-labeled oligopeptides to be used in receptor binding assays. 3H-Kyotorphin was prepared by this method at a yield of 72% and could be used in receptor binding assays after a single chromatographic separation.

The present review describes several lines of recent evidence providing new insights into the basic principles and mechanisms of axon projection from the olfactory epithelium to the olfactory bulb. Olfactory sensory neurons are classified into approximately 1000 subtypes according to the expression of specific odorant receptors. Olfactory sensory neurons expressing a given odorant receptor are distributed within one zone out of the four circumscribed zones of the olfactory epithelium and send their axons to the corresponding zone of the olfactory bulb: the principle of zone-to-zone projection. We discuss possible functions of a novel cell adhesion molecule, viz., OCAM, in the formation and maintenance of zone-to-zone projection of both olfactory and vomeronasal axons. Furthermore, olfactory sensory neurons expressing a given odorant receptor converge their axons onto only two topographically fixed glomeruli among the 1500-3000 glomeruli in the olfactory bulb: the principle of glomerular convergence. These axonal connection patterns give rise to the response specificity of the second-order neurons, viz., the mitral/tufted cells, to a particular range of odor molecules. In the process of glomerular convergence, combinatorial functions of axon-associated cell adhesion molecules and odorant receptor proteins may be required for the establishment of the precise targeting of olfactory axons to the appropriate glomeruli.

A device to control strength of interactions between adjacent nanostructures, namely Precise Interaction Control System for Nanostructures (PICSN) is developed. The PICSN is operated by combinations of DC motor and capacitancepositioning sensor. In this system, nanostructures are deposited on a flexible substrate and the strength of interactions can be controlled by changing a distance between adjacent nanostructures, stretching or shrinking the flexible substrate. Using the PICSN, we investigate photoluminescence of Au nanoclusters (NCs), which include at least Au5, Au8 and Au13 NCs, deposited on poly(dimethylsiloxane) substrate. We observed a blue shift of PL peak position with increasing interdot distance. Considering the relationship between absorption and emission energies of Au NCs, the blue shift is thought to be a result of fluorescent resonant energy transfer from smaller sized Au NCs to larger ones.