Simultaneous activities in both mirror-image glomerular maps in the olfactory bulb may have an important role in stress-related neuronal responses in mice.

In the mouse olfactory bulb (OB), odor input from the olfactory epithelium innervates topographically to form odorant maps, which are mirror-image arrangements of glomerular clusters with domain organization. However, the functional role of the mirror-image representation in the OB remains unknown. Predator odors induce stress responses, and the dorsal domain of the dorsolateral wall of the olfactory bulb (dlOB) is known to be involved in this process. However, it remains unclear whether the activities in the medial wall of the OB (mOB), the other mirror half, are also involved in stress responses. Therefore, in this study, we investigated whether the mOB and dlOB are required for the induction of stress responses using lesioning or electrical stimulation. Although there were no significant differences in the number of activated neurons in the bed nucleus of the stria terminalis, posterior piriform cortex or amygdalo-piriform transition area, fewer activated neurons were observed in the anterior piriform cortex (APC) following lesion of both the mOB and dlOB combined. No changes were observed in the density of activated cells in any examined brain region following stimulation of either the mOB or dlOB alone. However, activated neurons in the APC were significantly more numerous following simultaneous stimulation of the mOB and dlOB. Collectively, our results suggest that simultaneous activation in both the mOB and dlOB is needed to induce APC neural activities that produce stress-like behavior. These findings provide insight into olfactory information processing, and may also help in the development of therapies for odor-induced stress behaviors.

A Novel Monoclonal Antibody Against Neuroepithelial and Ependymal Cells and Characteristics of Its Positive Cells in Neurospheres.

There are still few useful cell membrane surface antigens suitable for identification and isolation of neural stem cells (NSCs). We generated a novel monoclonal antibody (mAb), designated as mAb against immature neural cell antigens (INCA mAb), which reacted with the areas around a lateral ventricle of a fetal cerebrum. INCA mAb specifically reacted with neuroepithelial cells in fetal cerebrums and ependymal cells in adult cerebrums. The recognition molecules were O-linked 40 and 42 kDa glycoproteins on the cell membrane surface (gp40 INCA and gp42 INCA). Based on expression pattern analysis of the recognition molecules in developing cerebrums, it was concluded that gp42 INCA was a stage-specific antigen expressed on undifferentiated neuroepithelial cells, while gp40 INCA was a cell lineage-specific antigen expressed at the stages of differentiation from neuroepithelial cells to ependymal cells. A flow cytometric analysis showed that fetal and young adult neurospheres were divided into INCA mAb(-) CD133 polyclonal antibody (pAb)(-), INCA mAb(+) CD133 pAb(-), and INCA mAb(+) CD133 pAb(+) cell populations based on the reactivity against INCA mAb and CD133 pAb. The proportion of cells having the neurosphere formation capability in the INCA mAb(+) CD133 pAb(+) cell population was significantly larger than that of undivided neurospheres. Neurospheres formed by clonal expansion of INCA mAb(+) CD133 pAb(+) cells gave rise to neurons and glial cells. INCA mAb will be a useful immunological probe in the study of NSCs.

Habitat odor can alleviate innate stress responses in mice.

Predatory odors, which can induce innate fear and stress responses in prey species, are frequently used in the development of animal models for several psychiatric diseases including post-traumatic stress disorder (PTSD) following a life-threatening event. We have previously shown that odors can be divided into at least three types; odors that act as (1) innate stressors, (2) as innate relaxants, or (3) have no innate effects on stress responses. Here, we attempted to verify whether an artificial odor, which had no innate effect on predatory odor-induced stress, could alleviate stress if experienced in early life as a habitat odor. In the current study, we demonstrated that the innate responses were changed to counteract stress following a postnatal experience. Moreover, we suggest that inhibitory circuits involved in stress-related neuronal networks and the concentrations of norepinephrine in the hippocampus may be crucial in alleviating stress induced by the predatory odor. Overall, these findings may be important for understanding the mechanisms involved in differential odor responses and also for the development of pharmacotherapeutic interventions that can alleviate stress in illnesses like PTSD.

Stress-related activities induced by predator odor may become indistinguishable by hinokitiol odor.

Predator odors, such as 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), induce a stress-like behavior in some rodents, and there is activation of a complex mix of brain regions including the anterior piriform cortex (APC) and the bed nucleus of stria terminalis (BST). In contrast, rose odor can counteract TMT-induced activation of the ventrorostral part of APC and the medial part of BST. In the present study, two novel odors, woody (hinokitiol) and caraway [S(+)-carvone] odors, were evaluated to determine whether they have an antistress effect. Plasma adrenocorticotropic hormone levels, a marker of stress, and the number of c-Fos-immunopositive cells were determined in APC and BST. Plasma adrenocorticotropic hormone levels were increased by TMT alone and in combination with S(+)-carvone; however, hinokitiol with or without TMT did not have an effect. The number of activated cells in the medial part of BST was increased by TMT alone and in combination with S(+)-carvone or hinokitiol. Although TMT alone activated the medial part of BST, a mixture of TMT and hinokitiol activated both the medial and the lateral part of BST. These data suggest that the selective responses to TMT in the medial part of BST were obscured by activation of more odor-related regions by hinokitiol with TMT. In addition, the ratio of medial to lateral BST activation may be critical in stress-related behavior. In conclusion, hinokitiol can alleviate TMT-induced stress; however, the underlying mechanism appears to be different from that of the rose odor, as found in our previous study.

Characterization of a monoclonal antibody, Namu mAb, which reacts to the subventricular zone in mouse brain.

The lateral ventricle in adult mammalian brain is widely acknowledged as one of the areas that undifferentiated neural cells such as neural stem cells and neural progenitor cells inhabit. However, immunological aspects of neural stem cells in the lateral ventricle are still under debate. Here, we report the generation and characterization of a novel monoclonal antibody (mAb), called Namu mAb, which stains the subventricular zone in the lateral ventricle of adult mouse brain. Namu mAb reacted to the cells in the subventricular zone and never reacted to differentiated neural cells such as neurons and glial cells such as astrocytes and oligodendrocytes. Its reaction pattern for the subventricular zone and the neurospheres was similar to that of Nestin and glial fibrillary acidic protein mAbs. Namu mAb recognition molecule, Namu antigen, was a 50 kDa protein present in the cytoplasmic fraction of mouse brain, and its expression was clearly observed in neurospheres cultured in the presence of epidermal growth factor, but it was never or only weakly induced in the presence of basic fibroblast growth factor or leukemia inhibitory factor. Collectively, it is concluded that Namu mAb specifically reacts to undifferentiated neural cells in mouse brain.

Rose odor can innately counteract predator odor.

When animals smell a predator odor such as 2,5-Dihydro-2,4,5-trimethylthiazoline (TMT), even if it is a novel substance, the hypothalamo-pituitary-adrenal (HPA) axis is activated, causing stress-like behaviors. Although the medial part of the bed nucleus of stria terminalis (mBST) is known to be involved in this process, the mechanism remains unclear. Moreover, it is unknown whether there is any odor that can counteract the predator odor, even when the odorants are novel substances for the animals. In this study, we assessed whether rose odor can counteract by counting the number of activated neurons in mice brain following the presentation of rose odor with or without TMT for 30 min. The number of activated cells in the mBST and in the ventrorostral part of the anterior piriform cortex (APC) was significantly reduced by a mixture of TMT and rose odor; however, no significant differences were noted in the dorsal part of the APC and in the olfactory bulb (OB) following TMT presentation with or without rose odor. The results suggest that rose odor may counteract the TMT-induced stress response in the OB and/or APC and suppress the neural circuit to the mBST. It also indicates that there are some odors that can innately counteract predator odor, even when they have not been experienced before.

Anatomical significance of a posterior horn of medial meniscus: the relationship between its radial tear and cartilage degradation of joint surface.

BACKGROUND: Traumatic injury and surgical meniscectomy of a medial meniscus are known to cause subsequent knee osteoarthritis. However, the difference in the prevalence of osteoarthritis caused by the individual type of the medial meniscal tear has not been elucidated. The aim of this study was to investigate what type of tear is predominantly responsible for the degradation of articular cartilage in the medial compartment of knee joints. METHODS: Five hundred and forty eight cadaveric knees (290 male and 258 female) were registered in this study. The average age of cadavers at death was 78.8 years old (range: 52-103 years). The knees were macroscopically examined and their medial menisci were classified into four groups according to types of tears: "no tear", "radial tear of posterior horn", "other types of tear" and "worn-out meniscus" groups. The severity of cartilage degradation in their medial compartment of knee joints was evaluated using the international cartilage repair society (ICRS) grading system. We statistically compared the ICRS grades among the groups using Mann-Whitney U test. RESULTS: The knees were assigned into the four groups: 416 "no tear" knees, 51 "radial tear of posterior horn" knees, 71 "other types of tear" knees, and 10 "worn-out meniscus" knees. The knees with substantial meniscal tears showed the severer ICRS grades of cartilage degradation than those without meniscal tears. In addition, the ICRS grades were significantly severer in the "radial tear of posterior horn" group than in the "other types of tear" group, suggesting that the radial tear of posterior horn in the medial meniscus is one of the risk factors for cartilage degradation of joint surface. CONCLUSIONS: We have clarified the relationship between the radial tear of posterior horn in the medial meniscus and the severer grade of cartilage degradation. This study indicates that the efforts should be made to restore the anatomical role of the posterior horn in keeping the hoop strain, when patients' physical activity levels are high and the tear pattern is simple enough to be securely sutured.

Flow cytometric analysis of mouse neurospheres based on the expression level of RANDAM-2.

RANDAM-2, a type-I transmembrane antigen constitutively expressed on the neuronal cell lineage during mouse neurogenesis, shows the highest expression level between embryonic day 8.5 (E8.5) and E10.5. As the period well overlaps with the proliferating stages of neural stem cells (NSCs), it is conceivable that NSCs are efficiently separable based on the expression level of RANDAM-2. In this paper, we show that NSCs can be efficiently enriched as RANDAM-2(high+) cells by fluorescence-activated cell sorting. Many cells in the RANDAM-2(high+) cells had the characteristics of the self-renewal capability and potential for multilineage differentiation into neural cells. In contrast, almost all of the RANDAM-2(low+/-) cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. These two subpopulations also differed from each other in terms of the expression level of molecules associated with neural differentiation. These findings demonstrate that RANDAM-2 can be regarded as a useful marker for enrichment of NSCs.

Clinical, biochemical, and cytochemical studies on a Japanese Salla disease case associated with a renal disorder.

We report the first Japanese case of Salla disease. A 5-year-old male patient developed unique proteinuria with other clinical manifestations, including coarse facies, dysostosis multiplex, mild mitral valve regurgitation, umbilical and inguinal herniation, and mild developmental delay. Pathological analysis of biopsied kidney tissues showed marked vacuolation of podocytes, mesangial cells, capillary endothelial cells, and tubular cells. Biochemical studies involving thin-layer chromatography and mass spectrometry revealed increased excretion of free sialic acid (N-acetylneuraminic acid) into the patient's urine. Immuno- and lectin staining of the patient's cells demonstrated the accumulation of sialyl and asialyl glycoconjugates in lysosomes and late endosomes. A defect in sialyl glycoconjugate metabolism is thought to have occurred in the patient's cells, besides impairment of the lysosomal transport of free sialic acid residues. A renal disorder should be considered as an important manifestation, not only in infantile free sialic acid storage disease but also in Salla disease.

Complementary DNA cloning and characterization of RANDAM-2, a type I membrane molecule specifically expressed on glutamatergic neuronal cells in the mouse cerebrum.

A membrane-surface glycoprotein, RANDAM-2, is one of the neuronal cell lineage-specific antigens involved in the neuronal differentiation of P19 embryonic carcinoma (EC) cells and the mouse central nervous system (CNS). Complementary DNA cloning of RANDAM-2 indicated that its nucleotide sequence completely matched that of PA2.26 antigen, a sialomucin-like transmembrane glycoprotein previously found on tumorigenic keratinocytes. RANDAM-2 transcripts were detectable from the embryonic stage of 6.5 days, and then the expression continued throughout the remaining embryonic stages and adulthood, with a localization restricted to the CNS. In growth factor-induced neurospheres and adult cerebrum, RANDAM-2-expressing cells coincided well not only with nestin-positive cells but also with glutamate-positive neurons, but not with gamma-aminobutyric acid-positive ones. These results indicate that RANDAM-2 is one of the type I membrane surface antigens constitutively expressed on undifferentiated neuronal cells and the glutamatergic neuronal cells during mouse neurogenesis.

Identification of neuronal cell lineage-specific molecules in the neuronal differentiation of P19 EC cells and mouse central nervous system.

P19 embryonic carcinoma (EC) cells are one of the simplest systems for analyzing the neuronal differentiation. To identify the membrane-associated molecules on the neuronal cells involved in the early neuronal differentiation in mice, we generated two monoclonal antibodies, SKY-1 and SKY-2, by immunizing rats with a membrane fraction of the neuronally committed P19 EC cells as an antigen. SKY-1 and SKY-2 recognized the carbohydrate moiety of a 90 kDa protein (RANDAM-1) and the polypeptide core of a 40 kDa protein (RANDAM-2), respectively. In the P19 EC cells, the expression of RANDAM-1 was colocalized to a part of Nestin-positive cells, whereas that of RANDAM-2 was observed in most Nestin-positive cells as well as beta-III-tubulin positive neurons. In the embryonic and adult brain of mice, RANDAM-1 was expressed at embryonic day 8.5 (E8.5), and the localization of antigen was restricted on the neuroepithelium and choroid plexus. The RANDAM-2 expression commenced at E6.0, and the antigen was distributed not only on the neuroepithelium of embryonic brain but on the neurons of adult brain. Collectively, it was concluded that RANDAM-1 is a stage specific antigen to express on the neural stem cells, and RANDAM-2 is constitutively expressed on both the neural stem cells and differentiated neuronal cells in mouse central nervous system (CNS).