Nobuhiko Miyasaka

Latexin, a carboxypeptidase A inhibitor, is expressed in rat peritoneal mast cells and is associated with granular structures distinct from secretory granules and lysosomes.

Latexin, a protein possessing inhibitory activity against rat carboxypeptidase A1 (CPA1) and CPA2, is expressed in a neuronal subset in the cerebral cortex and cells in other neural and non-neural tissues of rat. Although latexin also inhibits mast-cell CPA (MCCPA), the expression of latexin in rat mast cells has not previously been confirmed. In the present study we examined the expression and subcellular localization of latexin in rat peritoneal mast cells. Western blot and reverse-transcriptase-mediated PCR analyses showed that latexin was contained and expressed in the rat peritoneal mast cells. Immunocytochemically, latexin immunofluorescence was localized on granular structures distinct from MCCPA-, histamine- or cathepsin D-immunopositive granules. Immunoelectron microscopy revealed that latexin was associated with a minority population of granules. The latexin-associated granules were separated from MCCPA- or histamine-containing granules on a self-generating density gradient of polyvinylpyrrolidone-coated silica-gel particles (Percoll). Treatments with high ionic strength and heparinase released latexin from the granules, suggesting that latexin is non-covalently associated with a heparin-like component of the granules. MCCPA and histamine were released from the mast cells after non-immunological and immunological stimulation with compound 48/80, A23187 and anti-IgE antibody, whereas latexin was not released. These results show that latexin is synthesized in rat peritoneal mast cells and suggest that it is associated with a unique type of intracellular granules distinct from MCCPA- and histamine-containing secretory granules and lysosomes.

Foreign gene expression in an organotypic culture of cortical anlage after in vivo electroporation.

A high level of foreign gene expression in organotypic cultures of the cerebral cortical anlage was achieved by electroporation-mediated gene transfer in vivo. A mammalian expression plasmid for green fluorescent protein (GFP) gene was injected into the lateral ventricle of rat embryos. Immediately after the plasmid DNA injection, the head of the embryo was electroporated between a pair of tweezer-type electrodes. The cortical anlage was isolated and maintained organotypically up to 21 days in vitro (DIV). The GFP-transgene was expressed intensely in neural progenitor cells at 1 DIV. GFP-expressing cells were still detectable and were demonstrated to differentiate into neurons and glia at 21 DIV. This system is expected to be useful for molecular analysis of cerebral cortical development and function.

Olfactory receptor for prostaglandin F2[alpha] mediates male fish courtship behavior

Pheromones play vital roles for survival and reproduction in various organisms. In many fishes, prostaglandin F2α acts 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 F2α in zebrafish. Prostaglandin F2α specifically 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 F2α and impairment of courtship behaviors toward female fish. These findings demonstrate the functional significance and activation of selective neural circuitry for the sex pheromone prostaglandin F2α and its cognate olfactory receptor in fish reproductive behavior.

Comprehensive genetic analysis of zebrafish neural pathways from the olfactory bulb to higher brain centers

freezing behavior among a pair of odorant molecules, which had identical chemical compositions but had different steric structures. In our screening experiments, a majority of odorant receptors were commonly activated by both fear-odors and no-fear-odors, which have identical functional groups and have closely related chemical compositions. However, we have identified a small number of odorant receptors, which were activated by the multiple fear-odors, but not by no-fear-odors that had closely related chemical structures with the fear-odors. In this symposium, we will discuss about the relationships among fear-odors, fear-receptors, and fear responses. Mice can learn to associate an odorant molecule with an electrical shock to induce learned-freezing behavior. We compared physiological responses and neuronal activities in innate-freezing mice to those in learned-freezing mice. As a result, we have found that particular physiological responses and activation patterns of the neuronal circuits in the specific area in the brain were totally different between mice, which demonstrate innate and learned freezing behaviors. These results suggest that there are at least two distinct states of fear in the mice brain.

Genetic single-neuron tracing from the olfactory bulb to higher brain centers in zebrafish

We recently found that olfactory cortex generates sharp waves during slowwave sleep in freely behaving rats. Olfactory bulb also generates sharp wave-like activity, which is typically synchronized with olfactory cortex sharp waves. Here, we addressed the question which neuronal circuits generate olfactory cortex and olfactory bulb sharp waves using urethaneanesthetized rats.Olfactory cortex sharp waves occurred during slow-wave state in urethane-anesthetized rats. Many olfactory cortex neurons showed synchronous spike discharges at the descending phase of the olfactory cortex sharp waves. Current source density analysis indicated that olfactory cortex sharp waves were generated by recurrent association fiber synaptic inputs to pyramidal cells in anterior piriform cortex. Pyramidal neurons in the olfactory cortex give rise to massive centrifugal fibers that terminate on granule cells in the olfactory bulb. Sharp wave activity synchronized with olfactory cortex sharp waves occurred in the granule cell layer of the olfactory bulb during slow-wave state. Current source density analysis indicated that olfactory bulb sharp waves were generated by synchronous depolarization of proximal and basal dendrites of granule cells. These results suggest that olfactory cortex sharp waves travel to the granule cells in the olfactory bulb. Burst stimulation of the centrifugal fibers induced long lasting potentiation of the centrifugal fiber synapses on granule cells, suggesting that olfactory cortex sharp waves generate plastic changes in the synapses of granule cells.

Identification of olfactory sensory neurons that respond to alarm pheromone in zebrafish

Many fish species including zebrafish respond to putative alarm pheromone released from injured skin of conspecifics and display robust aversive behaviors characterized by burst swimming and freezing. This olfactory alarm response is thought to be an innate fear reaction, which would enable fish to evade predators. However, molecular, cellular, and neural-circuit mechanisms underlying the alarm response remain unknown. To identify neuronal populations activated by various physiological stimuli, several histochemical methods have been successfully used which detect the induction of immediate-early genes and the preceding phosphorylation of signal transduction machineries. In this study, we first found that the phosphorylation of Erk (MAPK kinase) can be used as a reliable marker for odor-induced activation of olfactory sensory neurons (OSNs) in zebrafish. For example, bile acids (putative social cues) induced phosphorylated Erk immunoreactivity (pErk-ir) in OSNs with long dendrites, whose cell bodies are located in the deep layer of the olfactory epithelium, whereas amino acids (feeding cues) induced pErk-ir in superficially located OSNs with short dendrites. These results are consistent with the previous reports that bile acids and amino acids activate ciliated and microvillous OSNs, respectively. We next exposed zebrafish to skin extract from conspecifics, which contain putative alarm pheromone. Strong pErk-ir was observed in pear-shaped OSNs lying close to the epithelial surface, and modest pErk-ir was detected in cells reminiscent of microvillous OSNs. Focusing on the pear-shaped OSNs that specifically respond to skin extract, further experiments are now in progress to clarify which olfactory receptors are expressed in this unique type of OSNs and how they project axons to the olfactory bulb to convey the information of alarm pheromone.

Neural circuit mechanism underlying olfactory alarm responses in zebrafish

Many fish species including zebrafish respond to putative alarm pheromone released from injured skin of conspecifics and display robust aversive behaviors characterized by burst swimming and freezing. This olfactory alarm response is thought to be an innate fear reaction, which would enable fish to evade predators. However, molecular, cellular, and neural-circuit mechanisms underlying the alarm response remain unknown. To identify neuronal populations activated by various physiological stimuli, several histochemical methods have been successfully used which detect the induction of immediate-early genes and the preceding phosphorylation of signal transduction machineries. In this study, we first found that the phosphorylation of Erk (MAPK kinase) can be used as a reliable marker for odor-induced activation of olfactory sensory neurons (OSNs) in zebrafish. For example, bile acids (putative social cues) induced phosphorylated Erk immunoreactivity (pErk-ir) in OSNs with long dendrites, whose cell bodies are located in the deep layer of the olfactory epithelium, whereas amino acids (feeding cues) induced pErk-ir in superficially located OSNs with short dendrites. These results are consistent with the previous reports that bile acids and amino acids activate ciliated and microvillous OSNs, respectively. We next exposed zebrafish to skin extract from conspecifics, which contain putative alarm pheromone. Strong pErk-ir was observed in pear-shaped OSNs lying close to the epithelial surface, and modest pErk-ir was detected in cells reminiscent of microvillous OSNs. Focusing on the pear-shaped OSNs that specifically respond to skin extract, further experiments are now in progress to clarify which olfactory receptors are expressed in this unique type of OSNs and how they project axons to the olfactory bulb to convey the information of alarm pheromone.

Colocalization of latexin with carboxypeptidase A in the infragranular neurons of cerebral cortex

Latexin is a carboxypeptidase A (CPA) inhibitor expressed in certain cortico-cortical projection neurons located in the infragranular layers of lateral cortical areas of adult rats and mice. To understand a physiological function of latexin in the cerebral cortex, we previously analyzed colocalization of latexin with protein phosphatase inhibitor-l and with Al-opioid receptor (K. Takiguchi-Hayashi & Y. Arimatsu, Neurosci. Res. Suppl. 21. S44, 1997). In the present study, we examined colocalization of latexin with CPA by immunohistochemistry. Restricted and similar distribution profiles were observed within the cerebral cortex for latexinand CPA-immunopositive neurons. In double immunofluorescence experiments in some cortical areas, it was shown that all of latexin-immunopositive neurons were also CPAimmunopositive. These observations suggest that latexin play a role together with CPA in the cortico-cortical circuit.

Analysis of regulatory region of the rat latexin gene

In mammals, two isozymes of topoisomerase II (Topo II) have been identified. Topo IIcl plays a key role in DNA replication. Physiological functions of Topo IIP are still unclear and its expression level is independent of cellular proliferation profiles. We have reported previously that expression of Topo IIP transiently increased in developing cerebellum. Analysis of genomic regions recognized by Topo I@ would certainly facilitate the understanding of its functional significance. To clone the genomic regions targeted by Topo I@, libraries were constructed from DNA fragments recovered by Topo IIP-specific antibodies from the rat cerebellum (10 days after birth) which had been incubated with VP16. Topo II inhibitors such as VP16 are well known to stabilize the covalent complex between Topo II and DNA (cleavable complex). Nucleotide sequencing of 42 clones at the both ends showed that two-thirds of the clones were multicopy repetitive sequences such as LINE (long interspersed nuclear sequence), satellite I and ID. The remaining one-third appeared to be single copy sequences which were derived mostly from noncoding regions. Detailed mapping of the Topo II cleavage sites is under progress.