A new role for photoresponsive neurons called simple photoreceptors in the sea slug Onchidium verruculatum: Potentiation of synaptic transmission and motor response.

The simple photoreceptors Ip-1 and Ip-2 are intrinsically light-sensitive neurons that exist in the abdominal ganglion of the sea slug Onchidium verruculatum. Using isolated ganglia and semi-intact or intact animal preparations, the present studies examined the light-sensing and physiological roles of Ip-1 and Ip-2 cells, which respond jointly to light by inducing a slow hyperpolarizing receptor potential. First, the synaptic inputs received by Ip-1 and Ip-2 and the axonal branches arising from their cell bodies were investigated. We found that these cells are not only first-order photosensory neurons, but also second-order neurons (interneurons), relaying inhibitory synaptic inputs such as water pressure and/or tactile senses. The amphibian Onchidium opens a pneumostome at low tide in order to aero-breathe. This pneumostome opening; i.e., aero-breathing behavior, was produced by spike discharges of Ip-1 and Ip-2 cells. Furthermore, the present results suggested that the hyperpolarizing photoresponse of Ip-1 and Ip-2 cells operates in the potentiation of inhibitory sensory synaptic transmission. Thus, we conclude that the simple photoreceptors of Onchidium play a role in the long-lasting potentiation of synaptic transmission and the subsequent behavioral response and so may be involved in a new photosensory modality, non-image-forming vision.

Retinopetal neurons located in the diencephalon of the Japanese monkey (Macaca fuscata).

After a monocular injection of the cholera toxin B subunit (CTB) into the vitreous chamber of one eye, the retrogradely labeled retinopetal neurons were studied in the diencephalon of the Japanese monkey. The retrogradely transported tracer was visualized using the peroxidase antibody technique and an anti-cholera toxin antibody. The CTB-labeled nerve cell bodies were scattered in the periventricular nucleus of the hypothalamus, lateral hypothalamic area, and midline nuclei of the thalamus on both sides. In addition, a few retrogradely labeled nerve somata were observed in the most rostral portion of the lateral geniculate nucleus on the contralateral side.

Effect of pre-treatment with St John's Wort on nephrotoxicity of cisplatin in rats.

An herbal health care supplement, St John's Wort (SJW, Hypericum perforatum) has become widely used in the treatment of depression, and is known to interact with therapeutic drugs. Here we report a preventive effect of SJW on cisplatin nephrotoxicity in rats. Rats were given SJW (400 mg/kg/day, p.o.) for 10 consecutive days, and were injected with cisplatin (5 mg/kg, i.v.) on the day after the final SJW treatment. Cisplatin treatment increased the serum creatinine level, which is an index of nephrotoxicity, to 1.51+/-0.22 mg/dl (mean+/-SE) from 0.28+/-0.05 mg/dl (control) on day 5 after the cisplatin injection. This increase fell significantly to 0.86+/-0.13 mg/dl by pre-treatment with SJW. Cisplatin-induced histological abnormality of the kidney was blocked by pre-treatment with SJW. When SJW was administered for 10 days, the amounts of renal metallothionein (MT) and hepatic multidrug resistance protein 2 (Mrp2) were increased to 164.8+/-13.0% and 220.8+/-39.3% (mean+/-SE) of controls, respectively. GSH levels in the kidney and liver were not changed. Total and free cisplatin concentration in serum was not influenced by SJW treatment. In conclusion, the results suggest that pre-treatment with SJW may diminish cisplatin nephrotoxicity.

Fasting-induced reduction in locomotor activity and reduced response of orexin neurons in carnitine-deficient mice.

We found reduced locomotor activity (LA) under fasting in systemic carnitine-deficient juvenile visceral steatosis (jvs(-/-)) mice. When food was withdrawn at 8:00 a.m. (lights-off at 7:00 p.m., 12h/cycle), the nocturnal LA of jvs(-/-) mice was much less than the control (jvs(+/+) and jvs(+/-)) mice. LA recovered under carnitine or sucrose administration, but not under medium-chain triglyceride. In addition, fasted jvs(-/-) mice, without any energy supply, were activated by modafinil, a stimulator of the dopamine pathway. These results suggest that the reduced LA is not adequately explained by energy deficit. As the fasted jvs(-/-) mice showed lower body core temperature (BT), we examined the central nervous system regulating LA and BT. We found lower percentage of c-Fos positive orexin neurons in the lateral hypothalamus and reduced orexin-A concentration in the cerebrospinal fluid of fasted jvs(-/-) mice. Sleep analysis revealed that fasted jvs(-/-) mice had disruption of prolonged wakefulness, with a higher frequency of brief episodes of non-REM sleep during the dark period than fasted jvs(+/+) mice. These results strongly suggest that the reduced LA in fasted jvs(-/-) mice is related to the inhibition of orexin neuronal activity.

Gender-specific amplification of Japanese macaque genes using primers for the human DYS391 and DYS438 loci.

The species specificity of 10 human Y chromosomal short tandem repeat loci, DYS19, DYS385, DYS389, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439, was examined in the Japanese macaque, Macaca fuscata. Primers for loci DYS391 and DYS438 only yielded an amplification product from male samples. The PCR products amplified with the DYS391 primers showed no inter-individual differences in migration rate in electrophoretic experiments. Sequence analysis revealed that these PCR products consisted of 287 bases containing tandem repeats of TC and TATC. The numbers of TC and the TATC repeats varied between individuals. The TC repeat does not exist in human and chimpanzee sequences. The PCR products amplified by the DYS438 primers provided no evidence of inter-individual variation between the six male Japanese macaque samples. In the Japanese macaque, PCR gives a 184 base pair product, in contrast to human sample from which the products are 203-233 bases in size. The primers for four loci, DYS19, DYS385, DYS389 and DYS437 produced PCR products from both male and female Japanese macaques. The primers for the other loci, DYS390, DYS392, DYS393 and DYS439, did not yield PCR products.

Identification of 16 novel mutations in the argininosuccinate synthetase gene and genotype-phenotype correlation in 38 classical citrullinemia patients.

Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1. ASS functions as a rate-limiting enzyme in the urea cycle. Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide. In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries. By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations. Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified. On the basis of primary sequence comparisons with the crystal structure of E. coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein. It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one). Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14. It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe. The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild. Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum. However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease.

Up-regulation of methionine-enkephalin-like immunoreactivity by 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment in the forebrain of the Long-Evans rat.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is considered to be one of the most toxic environmental contaminants, named dioxin. Exposure to TCDD induces a plethora of intoxication symptoms, including anorexia and hypothermia, in several mammals and human. Enkephalin, an endogenous pentapeptide, is an important neuroregulator of autonomic functions, such as food intake and body temperature. In this study, we investigated the effects of TCDD gastric administration on methionine-enkephalin (MEK) immunoreactivity in the brain of the Long-Evans rat, the species strain considered to be the most TCDD-susceptible, using immunohistochemical staining. A single dose of TCDD (dissolved in olive oil, 50 microg/kg) or olive oil alone was administrated to the rats by gavage. Compared with the vehicle-treated rat, a marked increase in the density of MEK immunoreactive cell bodies, fibers and terminals was found 2 weeks after TCDD treatment in the forebrain of the TCDD-treated rat, i.e. the central amygdaloid nucleus, field CA3 of the hippocampus, paraventricular hypothalamic nucleus, medial preoptic nucleus, interstitial nucleus of the posterior limb of the anterior commissure, lateral globus pallidus, ventral pallidum and lateral division of the bed nucleus of the stria terminalis. These results demonstrated for the first time a site-specific increased enkephalinergic activity in certain brain regions of the Long-Evans rat. It is suggested that the increased MEK immunoreactivity may act as a compensatory adaptation for the pathophysiological alterations caused by TCDD exposure.

Morphological study of orexin neurons in the hypothalamus of the Long-Evans rat, with special reference to co-expression of orexin and NADPH-diaphorase or nitric oxide synthase activities.

Orexins, novel neuropeptides, are exclusively localized in the hypothalamus and implicated in the regulation of a variety of activities, including food intake and energy balance. Nitric oxide (NO), an unconventional neurotransmitter, is widely present in numerous brain regions including the hypothalamus, and has similar physiological roles to those of the orexins. The present study was undertaken to examine the distribution of orexin neurons and the presence of neuronal nitric oxide synthase (nNOS) in the orexin neurons to clarify whether NO interacts with the orexins in the neuronal regulation activities in the Long-Evans rat. We used two double-labeling methods: nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry in combination with orexin immunohistochemistry, and double-labeling fluorescent immunohistochemistry for orexin and nNOS. The majority of the orexin immunoreactive neurons were localized mainly in the areas of the dorsomedial hypothalamic nucleus (DMN), the dorsal part of the perifornical nucleus (PEF) and lateral hypothalamic area. The orexin immunoreactive cell bodies were medium in size, and triangular, round, elliptic, and fusiform in shape. The sizes and shapes of orexin neurons in the different parts were similar. Cell bodies coexpressing the orexin and nNOS or NADPH-d were present in the areas of the DMN and the PEF, and the nerve fibers containing orexin and nNOS were distributed in the DMN and PEF, arcuate nucleus (ARN) and ventromedial hypothalamic nucleus (VMH). These results provide morphological evidence that there exists a population of nNOS- or NADPH-d-/orexin-coexpressing neurons in the orexinergic cell group in the hypothalamus, and taken together with previous findings, suggest that NO may play a role in the mechanisms by which orexin neurons regulate food intake and energy balance.

Effect of enucleation on the expression of c-Fos protein in the supraoptic nucleus of the Japanese monkey (Macaca fuscata).

The aim of this study was to examine the effects of one eye enucleation on the expression of c-Fos protein in the hypothalamus of the Japanese monkey (Macaca fuscata). Compared with an intact monkey, significantly increased numbers of c-Fos positive neurons were observed in the supraoptic nuclei on both sides at 1 h after eye enucleation. This maximal c-Fos expression then started to decrease at 3 h after eye enucleation. Furthermore, by a dual-labeled immunocytochemical study, the c-Fos immunoreactivity was found mainly in the vasopressinergic but not in the oxytocinergic neurons within the supraoptic nucleus. These results suggest that vasopressinergic but not oxytocinergic neurons within the supraoptic nucleus may have critical roles in the stimulation of this nucleus in response to eye enucleation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin treatment induces c-Fos expression in the forebrain of the Long-Evans rat.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most toxic environmental pollutants. In the present study, we examined c-Fos expression in the central nervous system (CNS) after administration of a lethal dose of TCDD to the adult Long-Evans rat to clarify if the CNS participates in TCDD-induced intoxication. A single dose of TCDD (dissolved in olive oil, 50 microg/kg) or olive oil alone was administered to the rats by gavage. Animals were allowed to survive for 1 day to 5 weeks. Three days after the administration, a significantly large number of Fos-immunopositive cells were found in the hypothalamus (i.e. dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus, medial preoptic nucleus), central amygdaloid nucleus and bed nucleus of the stria terminalis. These results suggest that some TCDD toxicity may be induced by its direct action on the CNS.

Role of the vestibular nuclei in endothelin-1-induced barrel rotation in rats.

The fourth or lateral ventricular injection of endothelin-1 resulted in a dose-dependent increase in the barrel rotation and produced marked induction of c-Fos-positive cells in the vestibular nuclei. The doses of the former injection were lower and had shorter mean latent periods compared with the later injection. c-Fos expression after endothelin-1 injection was prevented by the pretreatment with the endothelin ET(A) receptor antagonist, cyclo(D-alpha-aspartyl-L-propyl-D-valyl-L-leucyl-D-tryptophyl) (BQ-123), the glutamate NMDA receptor antagonist, dizocilpine maleate (MK-801), or the L-type Ca(2+) channel antagonist, verapamil, in addition to the incidence of the rotational behavior. There was a significant difference in c-Fos expression between the right and left medial vestibular nuclei, and the number of c-Fos-labeled neurons in the medial vestibular nucleus was markedly increased on the opposite side of the rotational direction. These results suggest that the elicitation of the barrel rotation may be mediated by endothelin ET(A) receptors, glutamate NMDA receptors, and L-type Ca(2+) channels. The changes in the receptor and channel systems induced by endothelin-1 injections appeared to exert crucial influences on the vestibular nuclei and then on the maintenance of equilibrium. The direction of the barrel rotation has a deep connection with the imbalance of neuronal activity in the left and right medial vestibular nuclei.

Dioxin exposure down-regulates nitric oxide synthase and NADPH-diaphorase activities in the hypothalamus of Long-Evans rat.

In this study, we investigated the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) gastric administration on the expression of neuronal nitric oxide synthase (nNOS) and the NADPH-diaphorase (NADPH-d) activities in the brain of the Long-Evans rat. A single dose of TCDD (dissolved in olive oil, 50 microg/kg) or olive oil alone was administered to the rats by gavage. nNOS Western blotting experiment indicated a marked decrease in nNOS immunoreactivity at 1 and 2 weeks after TCDD treatment. NADPH-d histochemistry results showed a marked decrease in the number of NADPH-d stained cell bodies in the paraventricular hypothalamic nucleus, lateral hypothalamic area and perifornical nucleus in the TCDD-treated rats. The present study suggests that TCDD administration down-regulates nitric oxide product in the hypothalamus, which may be partially responsible for TCDD-induced feeding inhibition.

Activation of hepatocyte growth factor in monkey stomach following gastric mucosal injury.

BACKGROUND: Hepatocyte growth factor (HGF) is involved in the proliferation and migration of various types of epithelial cells. HGF is produced as a single-chain precursor (pro-HGF) and functions after activation by HGF activator (HGFA). In this study, we aimed to examine the activation of pro-HGF to mature HGF and the expressions of HGF-related molecules, such as HGFA and HGFA inhibitor type 1 (HAI-1), in a monkey model of gastric mucosal injury. METHODS: Gastric mucosal injury was induced in Japanese monkeys by administration of HCl using nasal-gastric tubes. HGF activation was evaluated by Western blotting and enzyme-linked immunosorbent assay (ELISA), and the expression of HGF, HGFA, HAI-1, and c-Met were examined by Northern blotting and immunohistochemistry. RESULTS: Pro-HGF, but not mature HGF, was detected in normal gastric mucosa. When gastric mucosal injuries were induced, the expression of HGF significantly increased, and immunostaining of HGF was detected in fibroblasts in the gastric mucosa. In addition, conversion to mature HGF was observed in the injured gastric tissues, and mature HGF continued to increase for 48 h. HGFA was stably expressed in monkey livers, regardless of HCl administration. HAI-1 expression was detected in normal gastric mucosa, and its levels decreased after the induction of gastric mucosal injury. CONCLUSIONS: These results indicate that pro-HGF is stored in normal gastric tissues, and suggest that its conversion to mature HGF, associated with HGFA and HAI-1, is important for the repair of gastric mucosal injury.

Evaluation of the species specificity for six human short tandem repeat loci CSF1PO, TPOX, TH01, F13A01, FESFPS and vWA, in the Japanese macaque.

We examined six human short tandem repeat (STR) loci (c-fms proto-oncogene for CSF-1 receptor, CSF1PO; thyroid peroxidase, TPOX; tyrosine hydroxylase, TH01; coagulation factor XIII a subunit, F13A01; c-fes/fps proto-oncogene, FESFPS and von Willebrand factor, vWA) in the Japanese macaque, Macaca fuscata, using commercially available human STR kits. No products were amplified by polymerase chain reaction (PCR) using the human CSF1PO, TH01, FESFPS and vWA primers. Macaque DNAs were amplified with human TPOX and F13A01 primers. Macaque PCR products amplified with the TPOX and F13A01 primers migrated more slowly than human ones during electrophoresis. Sequencing results showed that the nucleotide sequences of the counterpart of the TPOX and F13A01 STR loci in the Japanese macaque were closely similar to those of the human genes except for tandem repeat regions. The macaque products amplified with human TPOX and F13A01 primers were highly polymorphic, with four variants of the former and 15 variants of the latter in the nine samples. These results indicate that the commercially available kits can be used to discriminate the Japanese macaque samples from human samples.

Polymerase chain reaction amplification of samples from Japanese monkeys using primers for human short tandem repeat loci.

We examined the species specificity of six commercially available human short tandem repeat systems (for CSF1PO, TPOX, TH01, F13A01, FESFPS and vWA) using six samples obtained from the Japanese monkey Macaca fuscata. Macaque genes were amplified with human TPOX and F13A01 primers. During electrophoresis, macaque polymerase chain reaction (PCR) products amplified with the TPOX primer migrated more slowly than human ones, and migrated close to human CSF1PO bands. Macaque PCR products amplified with the F13A01 primer also migrated more slowly than the human equivalent. The six macaque samples were classified into three types by the TPOX primer and into six types by the F13A01 primer. These results suggest that the primers for human TPOX and F13A01 loci can be used to distinguish between human and Japanese macaque samples.

Isolation and sequence analysis of the rat dihydrolipoamide succinyltransferase gene.

The dihydrolipoamide succinyltransferase (DLST) gene of the alpha-ketoglutarate dehydrogenase complex (alpha-KGDC) was isolated from a rat genomic DNA library and sequenced. This gene was composed of 15 exons and 14 introns like the human DLST gene. Sequence analysis of the promoter-regulatory region of the rat DLST gene-(Dlst) showed the possible presence of a CAAT box-sequence and of the sequences for an AP-2 site and three Sp1 sites, but no TATA box-sequence was evidenced. The nucleotide sequences of introns 1 and 4 of the rat Dlst were significantly homologous to those of introns 1 and 4 of the human DLST gene. The sequence analysis of the rat Dlst suggested that the exon coding for the E3- and/or E1-binding domain may have been lost from the gene during evolution in eukaryotic DLST, possibly after mitochondrial symbiosis because prokaryotic DLST possesses the E3- and/or E1-binding domain.

Do A-stretches inhibit the emergence of new variants in STR loci?

Penta E and D18S51 loci are known to be highly polymorphic in humans. In this study, Japanese macaque PCR products amplified with human Penta E and D18S51 primers were examined. Electrophoresis and sequence analyses revealed that 13 Japanese macaque products amplified with human Penta E primer were homogeneous. The Japanese macaque sequence was extremely similar with human Penta E alleles except for the insertion of contiguous adenosine repeats, called 'A-stretch', at 5'-side of AAAGA repeats. Among the 11 Japanese macaque PCR products amplified with human D18S51 primers, only two variants were observed. The sequences of these Japanese macaque products were similar to those of human D18S51 alleles. However, the Japanese macaque sequences also contained the insertion of A-stretch at 5'-side of AGAA repeats. Less polymorphism in the Japanese macaque sequences, in contrast with highly polymorphic human Penta E and D18S51 loci, suggested that A-stretches might inhibit the emergence of new variants at the STR loci.

Reduced N-acetylaspartate levels in mice lacking aralar, a brain- and muscle-type mitochondrial aspartate-glutamate carrier.

Aralar is a mitochondrial calcium-regulated aspartate-glutamate carrier mainly distributed in brain and skeletal muscle, involved in the transport of aspartate from mitochondria to cytosol, and in the transfer of cytosolic reducing equivalents into mitochondria as a member of the malate-aspartate NADH shuttle. In the present study, we describe the characteristics of aralar-deficient (Aralar-/-) mice, generated by a gene-trap method, showing no aralar mRNA and protein, and no detectable malate-aspartate shuttle activity in skeletal muscle and brain mitochondria. Aralar-/- mice were growth-retarded, exhibited generalized tremoring, and had pronounced motor coordination defects along with an impaired myelination in the central nervous system. Analysis of lipid components showed a marked decrease in the myelin lipid galactosyl cerebroside. The content of the myelin lipid precursor, N-acetylaspartate, and that of aspartate are drastically decreased in the brain of Aralar-/- mice. The defect in N-acetylaspartate production was also observed in cell extracts from primary neuronal cultures derived from Aralar-/- mouse embryos. These results show that aralar plays an important role in myelin formation by providing aspartate for the synthesis of N-acetylaspartate in neuronal cells.

Prolonged expression of c-Fos protein in the lateral habenular nucleus of the Japanese monkey (Macaca fuscata) after eye enucleation.

To elucidate the effect of traumatic stress on the lateral habenular nucleus, we investigated the time course of the expression of c-Fos protein in this nucleus of the Japanese monkey (Macaca fuscata) after enucleation of one eye using c-Fos protein immunocytochemistry. c-Fos protein-like immunoreactive neurons were significantly increased; the increase started 1 h after the enucleation and remained high for 3-9 h in the lateral habenular nucleus on both sides. These results suggest that the prolonged expression of c-Fos protein occurred in the lateral habenular nucleus after traumatic stress through multiple transsynaptic activations.