Mechanism of auditory hypersensitivity in human autism using autism model rats.

BACKGROUND: Auditory hypersensitivity is one of the major complications in autism spectrum disorder. The aim of this study was to investigate whether the auditory brain center is affected in autism model rats. METHODS: Autism model rats were prepared by prenatal exposure to thalidomide on embryonic day 9 and 10 in pregnant rats. The superior olivary complex (SOC), a complex of auditory nuclei, was immunostained with anti-calbindin d28k antibody at postnatal day 50. RESULTS: In autism model rats, SOC immunoreactivity was markedly decreased. Strength of immunostaining of SOC auditory fibers was also weak in autism model rats. Surprisingly, the size of the medial nucleus of trapezoid body, a nucleus exerting inhibitory function in SOC, was significantly decreased in autism model rats. CONCLUSIONS: Auditory hypersensitivity may be, in part, due to impairment of inhibitory processing by the auditory brain center.

Localization of the neuropeptide manserin in rat dorsal root ganglia: Involvement in nociceptive function.

Manserin, a neuropeptide discovered in the rat brain, is distributed in the spiral ganglion of the inner ear and carotid body, suggesting it is also localized in another neuron cluster. In this study, we examined manserin's localization in the dorsal root ganglion (DRG) and spinal cord of adult Wistar rats using immunohistochemical analyses. The DRG consists of neurofilament (NF) 200-positive large cells and two types of small cells (calcitonin gene-related peptide (CGRP)-positive peptidergic neurons and isolectin B4 (IB4)-positive non-peptidergic neurons). Manserin was localized in some of the small cells. Fluorescence double immunostaining showed that manserin-positive cells corresponded to some of the CGRP-positive cells. The DRG comprises pseudo-unipolar cells that receive sensory information from the skin and viscera and project to each layer of the dorsal horn of the spinal cord. Manserin was localized in the CGRP-positive layer I and II outer, but not in the IB4-positive layer II inner. These results suggest manserin is localized in CGRP-positive cells in the DRG, projects to the dorsal horn of the spinal cord, and is secreted with other neuropeptides, such as CGRP, to participate in nociceptive function.

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 (alpha cells), whereas they colocalized with insulin-secreting cells (beta cells) and somatostatin-secreting cells (delta 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.

Altered neuronal activity in the auditory brainstem following sound stimulation in thalidomide-induced autism model rats.

Auditory hypersensitivity in autism is frequently observed in clinics. Dysfunction in the auditory brainstem has been suspected. We have established autism model rats using prenatal thalidomide exposure. Here we investigated whether abnormal response occurs in the brainstem following sound stimulus in autism model rats. Autism model rats were prepared by prenatal exposure to thalidomide on embryonic days 9 and 10 in pregnant rats. Then, the animals were exposed to 16-kHz pure tone auditory stimulus and c-Fos immunostaining was performed to examine the neuronal activity on postnatal day 49 to 51. Following sound stimulus, increased number of c-Fos-positive neurons was observed in the medial nucleus of the trapezoid body of autism model rats compared with the control rats. These results suggest that prenatal thalidomide might cause altered processing of auditory stimulus, leading to the characteristics of auditory hypersensitivity in autism.

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.

Specific localization of manserin peptide in the rat carotid body.

The carotid body, located at the bifurcation of the common carotid artery, is a small sensory organ that detects changes in oxygen concentration and plays a vital role in controlling respiration. Although several molecules, such as neurotransmitters and neuropeptides, are involved in the regulation of the respiratory system, their detailed mechanisms have not been established yet. This study identifies that the presence of manserin, a neuropeptide, in the carotid body may play a crucial role in regulating respiration. The carotid bodies of adult Wistar rats were perfused with paraformaldehyde, and the frozen sections were subjected to immunohistochemical analyses. The carotid body comprises two distinct types of cells, neuron-like glomus cells and glial-like sustentacular cells. We used specific antibodies to distinguish the specific location of manserin in the carotid body, which included a tyrosine hydroxylase-positive antibody for glomus cells and an S100 protein antibody for sustentacular cells. Immunofluorescence analysis revealed that while tiny, round signals were exclusively observed in the cytoplasm of glomus cells, no signals were observed in sustentacular cells. Because manserin is believed to be secreted from precursor proteins by the endoproteolytic processing of a large precursor protein called secretogranin II, manserin secretion systems may exist in the carotid body, and thus, behave as potential regulators of respiration in the carotid body.

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.

[Genetic background of chronic fatigue syndrome].

Although previous twin and family studies have suggested the involvement of genetic factor(s) in the pathogenesis of chronic fatigue syndrome (CFS), responsible gene for CFS was not known. We have recently reported the association of serotonin transporter gene polymorphism in CFS. A significant increase of longer (L and XL) alleic variants was found in the CFS patients compared to the controls. Compared to S allele, the L allele is believed to retain higher transcriptional activity, which causes decreased concentration of serotonin in the extracellular space, namely, active serotonin in CFS. These results thus support the serotonin hypothesis in the pathogenesis of CFS.

Maternal administration of thalidomide or valproic acid causes abnormal serotonergic neurons in the offspring: implication for pathogenesis of autism.

Embryonic exposure to thalidomide (THAL) or valproic acid (VPA) before neural tube closure has been demonstrated as a useful model for human autism in rats. Abnormalities of the serotonergic system which are often observed in human autism have been shown in these rats. Thus, we examined whether early serotonergic neuronal development is perturbed by THAL/VPA. When pregnant rats were exposed to THAL or VPA on embryonic day 9, a dramatic shift of the distribution of serotonergic neurons in the dorsal raphe nucleus was observed on postnatal day 50. This alteration is thought to reflect abnormality of serotonergic neuronal differentiation and migration. In vitro studies revealed that VPA retards the maturation of serotonergic neuron from ES cell-derived neuronal progenitors, whereas exogenously added Sonic hedgehog, a morphogen that has been implicated in serotonergic cell fate, partially prevented this retardation. These results indicate that disruption of early serotonergic neuronal development might be involved in the etiology of autism.

Modification of AMPA receptor properties following environmental enrichment.

Environmental enrichment results in many modifications in the brain such as structural, behavioural, and biochemical changes. alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptors for excitatory amino acid glutamate are recently found to be involved in neuronal plasticity. In this study, we examined whether environmental enrichment modified the brain expression of mRNA for subunit composition of AMPA receptors in adult mice using the real-time quantitative PCR method and western blotting. Mice housed in enriched environments showed significantly higher levels of GluR2 and GluR4 subunits in the hippocampus compared to control mice. We concluded that environmental enrichment can change the expression of AMPA receptor subunits and thus might modify the potentials of brain plasticity.

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.

Cellular catabolism of lipid poor apolipoprotein E via cell surface LDL receptor-related protein.

Apolipoprotein E (apoE), an apoprotein involved in lipid transport in both the plasma and within the brain, mediates the binding of lipoproteins to members of the low density lipoprotein (LDL) receptor family including the LDL receptor and the LDL receptor-related protein (LRP). ApoE/LRP interactions may be particularly important in brain where both are expressed at high levels, and polymorphisms in the apoE and LRP genes have been linked to AD. To date, only apoE-enriched lipoproteins have been shown to be LRP ligands. To investigate further whether other, more lipid-poor forms of apoE interact with LRP, we tested whether lipid-free apoE in the absence of lipoprotein particles interacts with its cell-surface receptors. No detectable lipid was found associated with bacterially expressed and purified apoE either prior to or following incubation with cells when analyzed by electrospray ionization mass spectrometry. We found that the degradation of lipid-poor (125)I-apoE was significantly higher in wild type as compared to LRP-deficient cells, and was inhibited by receptor-associated protein (RAP). In contrast, (125)I-apoE-enriched beta-VLDL was degraded by both LRP and the LDL receptor. When analyzed via a single cycle of endocytosis, (125)I-apoE was internalized prior to its subsequent intracellular degradation with kinetics typical of receptor-mediated endocytosis. Thus, we conclude that a very lipid-poor form of apoE can be catabolized via cell surface LRP, suggesting that the conformation of apoE necessary for recognition by LRP can be imposed by situations other than an apoE-enriched lipoprotein.

Manserin, a novel peptide from secretogranin II in the neuroendocrine system.

We have isolated a novel 40 amino acid neuropeptide, designated manserin, from the rat. Manserin is derived from secretogranin II (SgII), a member of granin acidic secretory protein family by proteolytic processing, as previously reported secretoneurin and EM66. Immunohistochemical analysis using anti-manserin antibody revealed that manserin localized in the endocrine cells of the pituitary anterior lobe, but not in the posterior lobe. Interestingly, manserin never co-localized with ACTH in the anterior pituitary, which is in contrast with SgII, suggesting specific immunoreactivity of the antiserum against manserin. Manserin immunostaining was also observed in the neuronal cells of several hypothalamic nuclei and the neurons in the median eminence. These results suggest that manserin exerts a specific role in the neuroendocrine system.

Serum neurotrophin concentrations in autism and mental retardation: a pilot study.

To evaluate the availability of the serum neurotrophins for the diagnosis of the patients with neurodevelopmental disorder, we measured the serum concentration of brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) in the patients diagnosed with autism (n=18) and mental retardation (n=20), or healthy controls (n=16), using enzyme-linked immunosorbent assay. There tended to be a higher concentration of serum BDNF found in the autistic group ( P <0.05 by analysis of variance (ANOVA)) and the mental retardation group ( P <0.001 by ANOVA) compared to the control group. Serum NT-4 concentration tended to be increased in the mental retardation group (P <0.05 by ANOVA). We conclude that measuring the serum concentration of two neurotrophins, BDNF and NT-4, might be helpful to diagnose or classify disorders such as autism or mental retardation.

Localization of manserin, a secretogranin II-derived neuropeptide, in the oviduct of female rats.

Gynecological disorders related to menstrual cycle may be affected by stress and can cause infertility. Manserin is a stress-related neuropeptide that is present in the neuroendocrine system. In the present study, we determined the localization of manserin in the oviduct of adult Wistar rats using immunohistochemical techniques. Manserin was detected on the surface of the epithelium of the oviduct, but not in the ovary and uterus. Localization of manserin was specific to a large portion of the isthmus and to a small portion of the ampulla. These results suggest that manserin localizes to secretory cells in the oviduct and may be involved in stress-induced gynecological disorders.

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.

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.

Subtype-specific parafollicular localization of the neuropeptide manserin in the rat thyroid gland.

The thyroid gland is an endocrine organ which is involved in metabolism, neuroexcitability, body growth and development. The thyroid gland is also involved in the regulation of calcium metabolism, which is not yet fully understood. In this study, we investigated the localization of the granin-derived neuropeptide, manserin, in the adult rat thyroid gland. Manserin immunoreactivity was detected in thyroid follicular epithelial cells. Intense manserin signals were also detected in some, but not all, parafollicular cells, indicating that parafollicular manserin may be subtype-specific. These results indicate that thyroid manserin may play pivotal roles in parafollicular cells and follicular epithelial cells such as in calcium metabolism and/or thyroid hormone secretion.

Morphology of the facial motor nuclei in a rat model of autism during early development.

The development of facial nuclei in animal models of disease is poorly understood, but autism is sometimes associated with facial palsy. In the present study, to investigate migration of facial neurons and initial facial nucleus formation in an animal model of autism, rat embryos were treated with valproic acid (VPA) in utero at embryonic day (E) 9.5 and their facial nuclei were analyzed by in situ hybridization at E13.5, E14.5 and E15.5. Signals for Tbx20, which is expressed in early motor neurons, appeared near the floor plate at the level of the vestibular ganglion and extended caudolaterally, where they became ovoid in shape. This pattern of development was similar between control and VPA-exposed embryos. However, measurements of the migratory pathway and the size of the facial nuclei revealed that exposure to VPA hindered the caudal migration of neurons to the facial nuclei. Signals for cadherin 8, which is expressed in mature facial nuclei, revealed that exposure to VPA caused a significant reduction in the size of the facial nuclei. Our findings provide the first quantitative description of tangential migration and nucleus formation in the developing hindbrain in a rat model of autism.

Manserin as a novel histochemical neuroendocrine marker in prostate cancer.

OBJECTIVES: To investigate the presence of manserin in human prostate cancers and to correlate manserin expression with pathologic outcomes and progression-free survival. METHODS: Eighty-seven patients with recent prostate cancer were classified into 4 groups based on Gleason score, and manserin immunohistochemistry was correlated with Gleason sum grade. To investigate the validity of manserin as a prognostic factor, the Cox proportional hazards regression model was performed on 48 patients in our cohort with T3 or T4 prostate cancer who were initially treated with androgen deprivation therapy. RESULTS: The manserin-positive rates of patients with Gleason sums of 6, 7, 8, and ≥9 were 0%, 20.0%, 35.0%, and 48.1%, respectively. Manserin-positive rates were positively correlated with Gleason sums (P = 0.0001). Median times to cancer progression in groups with (n = 8) and without (n = 40) manserin expression were 8 months and 28 months, respectively (P = 0.01). Univariate Cox analysis revealed that manserin expression, clinical stage T4, and high Gleason sum were significantly associated with progression. Multivariate analysis revealed that only 2 factors, manserin expression (hazard ratio (HR) 4.99, P = 0.01) and clinical stage T4 (HR 4.77, P = 0.03), were independent risk factors for progression. CONCLUSIONS: This is the first report of manserin expression in human prostate cancers. Manserin may serve as a marker of prostate cancer progression.