Kayoko Hamaguchi-Hamada

Localization of 5-HT2A Receptor in rat cerebral cortex and olfactory system revealed by immunohistochemistry using two antibodies raised in rabbit and chicken

Serotonin 2A receptor (5-HT2A receptor) is widely distributed in the central nervous system, and has been suggested to be involved in a variety of behavioral conditions and neuropsychiatric disorders. Two polyclonal antibodies were raised against the N-terminus peptide of rat 5-HT2A receptor in chickens (5-HT2A-N) and a glutathione S-transferase fusion protein that contained the C-terminus of the mouse 5-HT2A receptor in rabbits (5-HT2A-C). Affinity-purified 5-HT2A-N and -C antibodies reacted strongly with a single band of 77-78 kDa in postsynaptic density proteins prepared from the rat cortex. The distribution pattern of immunoreactive structures in the rat brain was virtually the same for the two antibodies. The highest levels of immunoreactivity were observed in the olfactory bulb, neocortex, claustrum, piriform cortex, mamillary bodies, pontine nuclei, red nucleus and cranial motor nuclei. In the olfactory bulb, mitral cells were intensely labeled. In the neocortex, many immunoreactive neurons were found in layers II-VI. In layer IV of the neocortex, strong neuropil labeling was observed. In a double-labeling study using chicken 5-HT2A-N and rabbit anti-glial fibrillary acidic protein (GFAP) antibody, a considerable number of GFAP positive cells also showed 5-HT2A immunoreactivity. By using an immunoelectron microscopic technique, 5-HT2A receptor immunoreaction was shown to be localized just beneath the postsynaptic membrane thickening of asymmetric synapses.

Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer

When neural cells were collected from the entire cerebral cortex of developing mouse fetuses (15.5–17.5 days postcoitum) and their nuclei were transferred into enucleated oocytes, 5.5% of the reconstructed oocytes developed into normal offspring. This success rate was the highest among all previous mouse cloning experiments that used somatic cells. Forty-four percent of live embryos at 10.5 days postcoitum were morphologically normal when premature and early-postmitotic neural cells from the ventricular side of the cortex were used. In contrast, the majority (95%) of embryos were morphologically abnormal (including structural abnormalities in the neural tube) when postmitotic-differentiated neurons from the pial side of the cortex were used for cloning. Whereas 4.3% of embryos cloned with ventricular-side cells developed into healthy offspring, only 0.5% of those cloned with differentiated neurons in the pial side did so. These facts seem to suggest that the nuclei of neural cells in advanced stages of differentiation had lost their developmental totipotency. The underlying mechanism for this developmental limitation could be somatic DNA rearrangements in differentiating neural cells.

The cellular localization of 5-HT2A receptors in the spinal cord and spinal ganglia of the adult rat.

The localization of serotonin2A (5-HT2A) receptors in the adult rat spinal cord and dorsal root ganglia was examined by using a polyclonal antibody that recognizes the C-terminus peptides of the mouse 5-HT2A receptor. Positive cell bodies of 5-HT2A receptor were found in several regions of the spinal cord. Generally, large-to-intermediate sized neuronal cell bodies were intensely immunolabeled. Motoneurons in the ventral horn were the most intensely labeled. Dot-like immunoreactive profiles were located beneath the cell membrane of motoneurons. Neuronal somata in the intermediolateral nucleus of the thoracic spinal cord were moderately labeled. The immunoreactivity in the dorsal horn was weak. A considerable number of glial cell bodies in the white matter were immunostained. The majority of both small and large sized neurons were 5-HT2A immunopositive in the dorsal root ganglion.

Serotonin2A receptor-like immunoreactivity in rat cerebellar Purkinje cells

In the present study we examined the distribution pattern of serotonin2A (5-HT2A) receptors in the rat cerebellum. A strong immunoreaction against 5-HT2A receptor protein was observed in Purkinje cells. A dense cluster of immunopositive dendritic profiles of Purkinje cells was located beneath the pia matter of cerebellar cortex. Somal profiles in the cerebellar nuclei had weak to moderate immunoreactions.

Exposure to hexanal odor influences maternal behavior and induces neonatal death in Fyn tyrosine kinase-deficient mice.

Fyn-deficient pups born of Fyn-deficient parents die because they fail to suckle within 1-2 days after birth. Here we demonstrate that the neonatal death phenotype was influenced by the genetic background and an environmental odor. The odor of hexanal (C6-aldehyde) partially impaired mouse maternal behavior and induced the neonatal death of Fyn-deficient pups born of Fyn-deficient parents. This death phenotype was first observed in the breeding environment using autoclaved chips of Douglas fir. An analysis of the volatile chemicals in the autoclaved chips revealed an approximately 10-fold greater amount of hexanal than in non-autoclaved chips. Hexanal influenced the length of time virgin female mice engage in the maternal crouching behavior. In addition, Fyn-deficient females exhibited defects in the maternal behavior of nest building and pup retrieval, regardless of exposure to hexanal. These observations provide new insights into the regulation of maternal behavior by environmental and genetic factors.

Thiamine Deficiency Induces Massive Cell Death in the Olfactory Bulbs of Mice

Thiamine (vitamin B1) deficiency (TD) leads to focal brain necrosis in particular brain regions in humans and in experimental animal models. The precise mechanism of the selective topographic vulnerability triggered by TD still remains unclear. We examined the distribution pattern of cell death in the brains of mice in an experimental model of TD using anti-single-strand DNA immunohistochemistry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling methods. We found that interneurons in the olfactory bulb were sensitive to TD. The morphologic aspects of cell death in the olfactory bulb resembled those of cell death in thalamic neurons, which have previously been examined in detail. Furthermore, cell death in the olfactory bulb was partly relieved by the administration of an N-methyl-d-aspartate receptor antagonist, as was the case in thalamic lesions by TD. The superficial part of the olfactory granule cell layer seemed to be the most sensitive to TD, suggesting that differences in the afferents between superficial and deep granule cells may influence the sensitivity of these cells to TD. Our results indicate that the olfactory bulb should be considered as one of the vulnerable regions to TD.

Immunohistochemical evidence for the existence of novel mammalian neuropeptides related to the Hydra GLW-amide neuropeptide family

The GLW-amide family is a neuropeptide family found in cnidarian species and is characterized by the C-terminal amino acid sequence -Gly-Leu-Trp-NH2. To detect mammalian peptides structurally related to the GLW-amide family, we examined rat brain by immunohistochemistry with an anti-GLW-amide antibody. GLW-amide-like immunoreactivity (GLW-amide-LI) was observed in thin varicose fibers in some regions of the brain. Most neurons showing GLW-amide-LI were observed in the laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, and trigeminal/spinal ganglia. These results strongly suggest that the rat nervous system contains as yet unidentified GLW-amide-like peptides, and that GLW-amide-LI in the brain is a good marker for ascending projections from mesopontine cholinergic neurons.

Thrombospondin Type-1 Repeat Domain-Containing Proteins Are Strongly Expressed in the Head Region of Hydra

The head region of Hydra, the hypostome, is a key body part for developmental control and the nervous system. We herein examined genes specifically expressed in the head region of Hydra oligactis using suppression subtractive hybridization (SSH) cloning. A total of 1414 subtracted clones were sequenced and found to be derived from at least 540 different genes by BLASTN analyses. Approximately 25% of the subtracted clones had sequences encoding thrombospondin type-1 repeat (TSR) domains, and were derived from 17 genes. We identified 11 TSR domain-containing genes among the top 36 genes that were the most frequently detected in our SSH library. Whole-mount in situ hybridization analyses confirmed that at least 13 out of 17 TSR domain-containing genes were expressed in the hypostome of Hydra oligactis. The prominent expression of TSR domain-containing genes suggests that these genes play significant roles in the hypostome of Hydra oligactis.

Immunohistochemical evidence for the existence of novel mammalian neuropeptides related to the hydra GLWamide neuropeptide family

3UTR region of human dopamine transporter (DAT) gene contains variable number of tandem repeat (VNTR). VNTR has been thought to be associated with psychiatric disorders or personality. We previously identified HESR1 as a VNTR binding protein. Moreover, Hesr1 KO mice exhibited up-regulation of DAT and low spontaneous activity. In addition, we found that HESR family including HESR2 and 3, are also involved in DAT gene expression via VNTR using luciferase reporter assay. In the present study, we investigated localization of Hesr family in mice midbrain. Brain section was prepared for immunohistochemistory with anti-tyrosine hydroxylase and anti-Hesr1, 2 or 3 antibodies. We found that Hesr1 and 2 were localized in substantia nigra (SN) and ventral tegmental area (VTA). On the other hand, few Hesr3-positive cells were localized in SN or VTA. In addition, Hesr3 was mainly expressed in the cytoplasm while Hesr1 and 2 were in the nucleus.