Masaaki Narita

Nonexploratory movement and behavioral alterations in a thalidomide or valproic acid-induced autism model rat

Autism is a behaviorally characterized disorder with impairments in social interactions, as well as stereotyped, repetitive patterns of behaviors and interests. Exposure of rat fetuses to thalidomide (THAL) or valproic acid (VPA) on the ninth day of gestation has been reported as a useful model for human autism. We have shown that early serotonergic neural development is disrupted in these rats. In the current study, we used a radial maze and open field experimental paradigm to investigate whether these rats present behavioral and/or learning aberrations. THAL (500mg/kg), VPA (800mg/kg), or vehicle was administered orally to E9 pregnant rats at 7-10 weeks of age. Although the mean number of correct and incorrect arm choices in the initial eight arm choices did not differ between control and teratogen-exposed groups, achievement of learning (seven or eight consecutive correct choices for 3 consecutive days for individual rats) seemed to be impaired in teratogen-exposed groups. Interestingly, average time to explore the maze task was shorter in the teratogen-exposed groups, indicating that correct choice might be due to mere coincidence (i.e., nonexploratory movement). Unexpectedly, no significant differences were observed in social interaction in these rats. These results indicate that prenatal exposure to THAL and VPA might alter behavior in a manner that is, in part, consistent with human autism.

Maternal viral infection during pregnancy impairs development of fetal serotonergic neurons.

BACKGROUND Maternal viral infection during pregnancy induces morphological abnormalities in the fetus and may cause emotional and psychological problems in offspring through unknown mechanisms. We have previously shown that prenatal exposure of rats to chemicals such as thalidomide causes an autistic-like phenotype in offspring, indicating that prenatal events affecting serotonergic development may cause developmental disorder. METHODS We investigated whether prenatal viral infection altered the expression of neurotransmitters involved in the emotional or psychological status of offspring. We here took advantage of the polyriboinosinic:polyribocytidylic acid (poly I:C) system, the synthetic double-stranded RNA, which is often used in animal models of viral infection. RESULTS Ten mg/kg of poly I:C was intraperitoneally injected on gestational day (GD) 9 and counted the numbers of serotonin-immunopositive cells on GD15 using flat whole-mount preparation method, resulting 11.1% of increase in the number of serotonergic neurons in poly I:C group. Furthermore, there was a significant decrease in hippocampal serotonin content in offspring by postnatal day 50 following poly I:C administration by high-performance liquid chromatography. DISCUSSION AND CONCLUSION Since serotonin is known to link with behavior and emotion after birth, these results suggest that maternal viral infection might cause, in addition to morphological abnormalities, serotonin-related pathogenesis such as neurodevelopmental disorders including autism spectrum disorders.

Morphological abnormalities of embryonic cranial nerves after in utero exposure to valproic acid: implications for the pathogenesis of autism with multiple developmental anomalies

Autism is often associated with multiple developmental anomalies including asymmetric facial palsy. In order to establish the etiology of autism with facial palsy, research into developmental abnormalities of the peripheral facial nerves is necessary. In the present study, to investigate the development of peripheral cranial nerves for use in an animal model of autism, rat embryos were treated with valproic acid (VPA) in utero and their cranial nerves were visualized by immunostaining. Treatment with VPA after embryonic day 9 had a significant effect on the peripheral fibers of several cranial nerves. Following VPA treatment, immunoreactivity within the trigeminal, facial, glossopharyngeal and vagus nerves was significantly reduced. Additionally, abnormal axonal pathways were observed in the peripheral facial nerves. Thus, the morphology of several cranial nerves, including the facial nerve, can be affected by prenatal VPA exposure as early as E13. Our findings indicate that disruption of early facial nerve development is involved in the etiology of asymmetric facial palsy, and may suggest a link to the etiology of autism.

Recently identified a novel neuropeptide manserin colocalize with the TUNEL-positive cells in the top villi of the rat duodenum

We recently isolated a novel 40 amino acid neuropeptide designated manserin from the rat brain. Manserin is derived from secretogranin II, a member of granin acidic secretory protein family by proteolytic processing, as previously reported secretoneurin and EM66. Manserin peptide are localized in the endocrine cells of the pituitary. In this study, we further investigated the manserin localization in the digestive system by immunohistochemical analysis using antimanserin antibody. In the duodenum, manserin immunostaining was exclusively observed in the nuclei of top villi instead of cytosol as observed in neurons in our previous study. Interestingly, manserin‐positive cells in the duodenum are colocalized with terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) positive cells, the cells whose DNA was damaged. Since the top villi of duodenum epithelial cells are known to undergo spontaneous apoptosis during epithelial cell turn over, and since other peptides such as secretoneurin and EM66 derived from SgII have been reported to be cancer‐related, these results indicated that manserin peptide may have a role in apoptosis and/or cancer pathogenesis in the digestive organ. Copyright

Neuropathological alteration of aquaporin 1 immunoreactive enteric neurons in the streptozotocin-induced diabetic rats

Long-term diabetic patients exhibit major clinical gastrointestinal problems, such as diarrhea and constipation. In recent years, water channel protein, aquaporin 1 (AQP1) has been identified in the enteric nervous system (ENS). We have examined the pathological changes in AQP1 immunoreactive (IR) neurons in streptozotocin-induced (STZ) diabetic rats. Eight-week-old Wistar rats were injected with streptozotocin, and artificial diabetes was induced. Sixteen-week-old STZ rats were then examined with double immunofluorescence staining and ABC immunohistochemical staining. AQP1-IR neurons in STZ rats were significantly increased compared with control rats (p<0.01). The ratio of AQP1 vs. HuC/D in STZ rats was also clearly increased as compared with control rats (p<0.05). It was apparent that thick AQP1-IR fibers were frequently observed in the secondary and tertiary myenteric plexus of STZ rats. The AQP1-IR fibers of STZ rats conspicuously showed many swollen varicosities. These swollen varicose fibers were also observed in the longitudinal and circular muscle layers. Streptozotocin-induced diabetic rats showed pathological changes in AQP1-IR neurons of the ENS. The alteration of AQP1-IR neurons may be possible contribute to diabetic gastrointestinal dysfunction in streptozotocin-induced diabetic rats.

Prenatal exposure to organomercury, thimerosal, persistently impairs the serotonergic and dopaminergic systems in the rat brain: implications for association with developmental disorders.

Thimerosal, an organomercury compound, has been widely used as a preservative. Therefore, concerns have been raised about its neurotoxicity. We recently demonstrated perturbation of early serotonergic development by prenatal exposure to thimerosal (Ida-Eto et al. (2011) [11]). Here, we investigated whether prenatal thimerosal exposure causes persistent impairment after birth. Analysis on postnatal day 50 showed significant increase in hippocampal serotonin following thimerosal administration on embryonic day 9. Furthermore, not only serotonin, striatal dopamine was significantly increased. These results indicate that embryonic exposure to thimerosal produces lasting impairment of brain monoaminergic system, and thus every effort should be made to avoid the use of thimerosal.

Embryonic exposure to thimerosal, an organomercury compound, causes abnormal early development of serotonergic neurons

Even though neuronal toxicity due to organomercury compounds is well known, thimerosal, an organomercury compound, is widely used in pediatric vaccine preservation. In the present study, we examined whether embryonic exposure to thimerosal affects early development of serotonergic neurons. Thimerosal (1mg Hg/kg) was intramuscularly administered to pregnant rats on gestational day 9 (susceptible time window for development of fetal serotonergic system), and fetal serotonergic neurons were assessed at embryonic day 15 using anti-serotonin antibodies. A dramatic increase in the number of serotonergic neurons localized to the lateral portion of the caudal raphe was observed in thimerosal group (1.9-fold increase, p<0.01 compared to control). These results indicate that embryonic exposure to thimerosal affects early development of serotonergic neurons.

The effects of prenatal exposure to valproic acid on the initial development of serotonergic neurons.

In utero exposure to valproic acid (VPA) may cause symptoms related to autism spectrum disorder (ASD). An abnormal serotonergic (5‐HT) system has been implicated in the etiology of ASD. In the present study, we have examined the expression and distribution of two early inducers of 5‐HT neurons in rat embryos, to elucidate the prenatal development of 5‐HT neurons after VPA exposure at embryonic day (E) 9.5. Whole‐embryo in situ hybridization at E11.5 showed that the expression of sonic hedgehog, one of the early inducers of 5‐HT neurons, was reduced around the isthmus in the VPA‐exposed group. Furthermore, whole‐mount immunohistochemistry of the hindbrain and quantitative analysis of 5‐HT neurons in the rostral raphe nucleus (rRN) revealed that neuronal distribution in the caudal part of the rRN was narrower at E15.5 in the VPA‐exposed group than in controls. Thus, the early development of 5‐HT neurons was altered after VPA exposure in utero. The observed prenatal alteration may be significant in the etiology of autism.

Hyperkalemia and hyperdopaminemia induced by an obsessive eating of banana in an anorexia nervosa adolescent

Banana is known as a dopamine-rich and potassium-rich food, however no previous data regarding biochemical or psychological alteration induced by excess intake of banana has been reported. We have experienced an adolescent female case of Anorexia nervosa (AN) who denied eating anything but maximum 20 bananas and less than 500 ml mineral water per day for more than two years. During the period of massive banana eating habit, she showed increase of serum potassium (from 4.7 mEq/l to 6.1 mEq/l) and whole blood dopamine (from 11 ng/ml to 210 ng/ml; normal range 0.5-6.2 ng/ml), and obvious dysthymia that is inexplicable only by the pathology of AN. When the patient resumed other food ingestion after 26 months of obsessive and restricted eating of banana, the abnormalities in her blood data and her psychological state were all corrected toward normal. We conclude that in this case, the obsessive and restricted habit of banana ingestion resulted in hyperkalemia, hyperdopaminemia, and psychological change.

Manserin, a secretogranin II-derived peptide, distributes in the rat endocrine pancreas colocalized with islet-cell specific manner

Manserin is a recently characterized 40-amino acid neuropeptide derived from secretogranin II, a protein belonging to the chromogranin family. Although the physiological roles of manserin have not been elucidated to date, manserin has been shown to distribute in not only the brain but also the endocrine system such as the pituitary and adrenal glands, suggesting its role in the endocrine system. The present study aimed to explore the occurrence and distribution of manserin in the rat pancreas using an immunohistochemical technique with a polyclonal antibody against rat manserin. Immunoreactivity for manserin was readily detected in almost whole islets of Langerhans whereas not at all in the exocrine pancreas. Manserin-expressing cells were not colocalized with the glucagon-secreting cells (α cells), whereas they colocalized with insulin-secreting cells (β cells) and somatostatin-secreting cells (δ cells), although their intracellular distribution was different. These results indicate that manserin, occurring in the endocrine pancreas, may have a potential role in the endocrine system.