Chiaki Masuda

Distribution of natriuretic peptide receptor-C immunoreactivity in the rat brainstem and its relationship to cholinergic and catecholaminergic neurons

The natriuretic peptide receptor type C (NPR-C) binds all natriuretic peptides. It is thought to be involved in the clearance of natriuretic peptides and more recently has been defined as essential for the neuromodulatory effects of natriuretic peptides. Although the distribution of NPR-C mRNA has been reported in the rat forebrain, there are no data on the distribution of NPR-C in the brainstem. We report an immunofluorescence study on the distribution of NPR-C immunoreactivity in the rat brainstem, and its presence in cholinergic and catecholaminergic neurons. NPR-C immunoreactivity was detected in several regions, including the periaqueductal gray, oculomotor nucleus, red nucleus and trochlear nucleus of the midbrain; the pontine nucleus, dorsal tegmental nucleus, vestibular nucleus, locus coeruleus, trigeminal motor nucleus, nucleus of the trapezoid body, abducens nucleus and facial nucleus of the pons; and the dorsal motor nucleus of the vagus, hypoglossal nucleus, lateral reticular nucleus, nucleus ambiguus and inferior olivary nucleus of the medulla oblongata. Interestingly, NPR-C immunoreactivity was detected in the cholinergic neurons of the oculomotor nucleus, trochlear nucleus, dorsal tegmental nucleus, motor trigeminal nucleus, facial nucleus, dorsal motor nucleus of the vagus, nucleus ambiguus and hypoglossal nucleus. Furthermore, NPR-C immunoreactivity was detected in several catecholaminergic neuronal groups including the A6, A5, A1, C3 and C1 cell groups. These results are consistent with an important role for natriuretic peptides in neuroendocrine regulation and central cardiovascular integration. The extensive distribution of NPR-C in the brainstem supports the hypothesis that NPR-C is involved in the neuromodulatory effect of natriuretic peptides.

Expression of natriuretic peptide-activated guanylate cyclases by cholinergic and dopaminergic amacrine cells of the rat retina.

Recently, the natriuretic peptides were detected in the cholinergic and dopaminergic amacrine cells of the retina. We performed immunofluorescence labeling of rat retinal sections to examine the immunoreactivity of natriuretic peptide-activated guanylate cyclases (NPR-A and NPR-B) in the rat retina, in particular whether they were localized to dopaminergic and cholinergic amacrine cells. NPR-A and NPR-B immunoreactivity was detected in several layers of the retina including amacrine cells. In amacrine cells, both NPR-A and NPR-B were co-localized with tyrosine hydroxylase, a marker of dopaminergic cells. NPR-B, but not NPR-A, was localized to amacrine cells expressing choline acetyltransferase (ChAT), a marker of cholinergic cells. These findings suggest that natriuretic peptides have different regulatory systems in dopaminergic and cholinergic amacrine cells in rat retina.

Immunohistochemical Mapping of TRK-Fused Gene Products in the Rat Brainstem

The TRK-fused gene (TFG in human, Tfg in rat) was originally identified in human papillary thyroid cancer as a chimeric form of the NTRK1 gene. It was since reported that the gene product (TFG) plays a role in regulating phosphotyrosine-specific phosphatase-1 activity. As shown in the accompanying paper, we produced an antibody to rat TFG and used it to localize TFG to selected neurons in specific regions. In the present study, we mapped the TFG-positive neurons in the brainstem, cerebellum, and spinal cord of rats. In the brainstem, neurons intensely positive for TFG were distributed in the raphe nuclei, the gigantocellular reticular nucleus, the reticulotegmental nucleus of the pons, and some cranial nerve nuclei such as the trigeminal nuclei, the vestibulocochlear nuclei, and the dorsal motor nucleus of the vagus. Purkinje cells in the cerebellum and motor neurons in the spinal anterior horn were also positive for TFG. These results provide fundamental data for studying the functions of TFG in the brain.

MR tracking of transplanted glial cells using poly-l-lysine-CF3

Magnetic resonance (MR) imaging using super-paramagnetic iron oxides (SPIOs) is a powerful tool to monitor transplanted cells in living animals. Since, however, SPIOs are negative contrast agents, positive agents have been explored. In this study, we examined the feasibility of FITC-labeled poly-L-lysine-CF3 (PLK-CF3) using glial cells. FITC-labeled PLK-CF3 was easily internalized by neuroblastoma cells and glia as adding it into culture medium. No toxicity was seen at the concentration of less than 80 microg/ml. MR images positively detected labeled cells transplanted in the brain of living mouse. The results indicate that FITC-labeled PLK-CF3 is a useful positive contrast agent for MR tracking.

Expression and Localization of TRK-Fused Gene Products in the Rat Brain and Retina

The TRK-fused gene (TFG in human, Tfg in rat) was originally identified in human papillary thyroid cancer as a chimeric form of the NTRK1 gene. It has been reported that the gene product (TFG) plays a role in regulating phosphotyrosine-specific phosphatase-1 activity. However, no information regarding the localization of Tfg in rat tissues is available. In this study, we investigated the expression of Tfg mRNA in normal rat tissues using reverse transcription-polymerase chain reaction (RT-PCR). We also produced an antibody against Tfg gene products and examined the localization of TFG in the rat brain and retina. The RT-PCR experiments demonstrated that two types of Tfg mRNA were expressed in rat tissues: the conventional form of Tfg (cTfg) and a novel variant form, retinal Tfg (rTfg). RT-PCR analyses demonstrated that cTfg was ubiquitously expressed in rat tissues, while rTfg was predominantly expressed in the brain and retina. Western blot analysis demonstrated two bands with molecular weights of about 30 kDa and 50 kDa in the rat brain. Immunohistochemistry indicated that TFG proteins were predominantly expressed by neurons in the brain. In the rat retina, intense TFG-immunoreactivity was detected in the layer of rods and cones and the outer plexiform layer.

The mRNA Distribution of C7orf24, a γ-Glutamyl Cyclotransferase, in Rat Tissues

The putative protein C7orf24 is encoded by Homo sapiens chromosome 7 open reading frame 24. C7orf24 was first identified as a 21-kDa cytochrome c-releasing factor detected in the cytosolic fraction of human leukemia U937 cells after treatment with geranyl-geraniol. C7orf24 protein was recently identified as a γ-glutamyl cyclotransferase, an enzyme in the

Immunohistochemical localization of an isoform of TRK-fused gene-like protein in the rat retina

gL-glutamyl cycle. However, the exact localization of C7orf24 mRNA in normal tissues remains unknown. The present study examined the distribution pattern of C7orf24 mRNA in rat tissues using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization histochemistry. The RT-PCR experiments demonstrated that C7orf24 and a variant C7orf24 mRNA were expressed in various tissues. Quantitative RT-PCR analysis revealed significantly high levels of both C7orf24 mRNAs in the liver and kidney, compared with other tissues examined. In situ hybridization histochemistry localized C7orf24 mRNA to hepatocytes in the liver and renal tubules in the kidney. The present results thus implicated an important role for C7orf24 in liver and kidney. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Striatal and extrastriatal dopamine release in the common marmoset brain measured by positron emission tomography and [18F]fallypride

The TRK-fused gene (TFG) was originally identified in chromosome translocation events, creating a pair of oncogenes in some cancers, and was recently demonstrated as the causal gene of hereditary motor and sensory neuropathy with proximal dominant involvement. Recently, we cloned an alternative splicing variant of Tfg from a cDNA library of the rat retina, tentatively naming it retinal Tfg (rTfg). Although the common form of Tfg is ubiquitously expressed in most rat tissues, rTfg expression is localized to the central nervous system. In this study, we produced an antibody against an rTFG-specific amino acid sequence and used it to examine the localization of rTFG-like protein in the rat retina by immunohistochemistry and Western blots. Western blot analysis showed that the antibody detected a single band of 24 kDa in the rat retina. When we examined rTFG recombinant protein, the antibody detected two bands of about 42 kDa and 24 kDa. The results suggest that the 24 kDa rTFG-like protein is a fragment of rTFG. In our immunohistochemical studies of the rat retina, rTFG-like immunoreactivity was observed in all calbindin D-28K-positive horizontal cells and in some syntaxin 1-positive amacrine cells (ACs). In addition, the rTFG-like immunopositive ACs were actually glycine transporter 1-positive glycinergic or glutamate decarboxylase-positive GABAergic ACs. Our findings indicate that this novel 24 kDa rTFG-like protein may play a specific role in retinal inhibitory interneurons.

357. Blood-Brain Interface Opening By Ultrasound in Adult Common Marmoset To Induce Brain Pathology With rAAV

Previous studies have demonstrated that patients with schizophrenia show greater sensitivity to psychostimulants than healthy subjects. Sensitization to psychostimulants and resultant alteration of dopaminergic neurotransmission in rodents has been suggested as a useful model of schizophrenia. This study sought to examine the use of methylphenidate as a psychostimulant to induce dopamine release and that of [(18)F]fallypride as a radioligand to quantify the release in a primate model of schizophrenia. Four common marmosets were scanned by positron emission tomography twice, before and after methylphenidate challenge, to evaluate dopamine release. Four other marmosets were sensitized by repeated methamphetamine (MAP) administration. Then, they were scanned twice, before and after methylphenidate challenge, to evaluate whether MAP-sensitization induced greater sensitivity to methylphenidate. We revealed a main effect of the methylphenidate challenge but not the MAP pretreatment on the striatal binding potential. These results suggest that methylphenidate-induced striatal dopamine release in the common marmoset could be evaluated by [(18)F]fallypride.

The MR tracking of transplanted ATDC5 cells using fluorinated poly-l-lysine-CF3

Background: The common marmoset (Callithrix jacchus) could provide an appropriate model for neuromuscular diseases because of its higher brain function and physiological resemblance to human. Although production of transgenic marmosets from zygote transduced with lentiviral vector were reported, this strategy is too costly and not efficient with various reasons. In contrast, an adeno-associated virus vector (rAAV) has great advantages to induce pathology in the aged animals. However, fully maturated blood-brain interface (BBI) significantly limits passive rAAV transport from circulation to the brain. To overcome this issue, we investigated BBI opening, which is a promising technology to make cerebral capillary open by transiently disrupting tight junction in BBI with ultrasound irradiation (UI) just after i.v. injection of microbubble (MB).Methods: Commercially available MB or homemade bubbleliposome (BL) mixed with evans blue were i.v. injected into the femoral vein of the anesthetized adult marmosets. rAAV1 was also injected simultanously in certain trials. Subsequently, transcranial (TC) UI to the brain was performed for several minutes. The brain was removed 24 hrs after the UI. To visualize the leakage of albumin (ALB) in more detail, cryosections were immunologically stained with antibodies against ALB and CD31. Thin coronal slice of the brain was pieced into various parts, and genome DNA was extracted from the each pieces. Then relative rAAV genome copies were measured by qPCR. For live imaging analysis, BL mixed with carbon 11-labeled aminoisobutyric acid were i.v. injected into the tail vein. Subsequently, TCUI and positron emission tomography (PET) scan were performed.Results: In TCUI from the temporal region, ALB mainly leaked at the temporal cortex and hippocampus. In TCUI from circumference, ALB leakage seemed to occur at whole brain except for white matter.Microscopically, ALB leakage was found around the blood vessels of 10-50 mm in diameter. Especially, leakage in hippocampus was prominent. Relative rAAV genome copies were imbalanced between symmetrical piece pair. PET revealed ALB leakage not only in hippocampus but also in the anterior cingulate cortex, basal ganglia, optic tract, amygdala, and superior temporal gyrus.Conclusion: UI-mediated MB-assisted TC-BBI opening is promising approach in the generation of the disease model based on adult common marmoset.