FK506-protective effects against trimethyltin neurotoxicity in rats: hippocampal expression analyses reveal the involvement of periarterial osteopontin.

There is little information on the molecular mechanisms in FK506-mediated neuroprotection. In the present study, we investigated the protective effect of FK506, an immunosuppressant and neuroprotectant, on trimethyltin (TMT)-induced neurotoxicity in the rat hippocampus. Histologically, TMT-induced neuronal damage was partially prevented by FK506 in the hippocampal CA1 region, but not in CA3. FK506 treatment significantly reduced the number of apoptotic cells in CA1, but not in CA3, and also prevented induction of cognitive deficits by TMT. Microarray analysis of the rat hippocampus detected 14 genes with TMT-induced alteration of mRNA expression that was rescued by FK506 treatment. Subsequent quantitative RT-PCR analysis confirmed elevated mRNA levels for four inflammatory genes, glutathione S-transferase, lysozyme, matrix Gla protein, and osteopontin after TMT treatment. Upregulation of these genes was reversed by FK506 treatment at 5 days postgavage. Immunohistochemistry revealed that FK506 reduced osteopontin (OPN) induction by TMT in the periarterial area at 5 days postgavage. Our data suggest that inflammatory gene expression is involved in TMT-induced damage to the hippocampal CA1 region, resulting in apoptosis, and that this process is initiated by periarterial OPN activation, and can be alleviated by FK506.

Perinatal bisphenol A affects the behavior and SRC-1 expression of male pups but does not influence on the thyroid hormone receptors and its responsive gene.

Bisphenol A (BPA) has been shown to interfere with thyroid hormone receptors (THRs) and to influence the expression of THR-responsive elements in vivo and in vitro, while some studies reported hyperactivity induced by BPA treatment. In the present study, our purpose was to investigate the effect of BPA exposure on behavioral alteration and its mechanism of action, especially focusing on the thyroid hormone pathway. Significant sexual difference on behaviors was observed in perinatal BPA exposure, as manifested by hyperactivity and impaired spatial learning/memory in male pups after matured. Dams treated with 0.1mg/l BPA showed transient hypothyroidism, while male pups were found to exhibit a transient hyperthyroidism followed by hypothyroidism. Furthermore, significant up-regulated expression levels of mRNA and protein of SRC-1 in the hippocampus were observed in male pups by 0.1mg/l BPA treatment. However the expression of THRalpha/beta and RC3/neurogranin were not affected by BPA treatment. These results indicate that perinatal BPA exposure at a very low level may influence thyroid function and then consequently affects brain development, but at the same time, suggest that thyroid hormone receptor may not be a direct target of BPA action, but instead, another factor may be involved in this action.

Cytokines participate in neuronal death induced by trimethyltin in the rat hippocampus via type II glucocorticoid receptors.

We investigated the role of IL-1alpha and IL-1beta expressed in the reactive gliosis following hippocampal damage induced by trimethyltin (TMT). IL-1alpha immunoreactivity was expressed earlier in small glial cells on day 4 post-TMT, while IL-1beta expression was obvious in large swollen glial cells on day 14 post-TMT. Both IL-1alpha and IL-1beta immunoreactivities were double-labeled with astrocyte marker, vimentin, but not with a microglia marker, OX-42. The expression of both IL-1alpha/beta was enhanced by adrenalectomy (ADX) prior to TMT administration. Corticosterone (CORT) or dexamethasone (DEX) supplementation not only cancelled effects of ADX, but also partially reversed TMT-induced enhancement of IL-1alpha/beta expressions. These changes coincided with TMT-induced neuronal death in CA3 pyramidal cells of the hippocampus. It is suggested that IL-1alpha/beta expressed in reactive astrocytes participate in TMT neurotoxicity via type II glucocorticoid receptors.

Cytokines participate in neuronal death induced by trimethyltin in the rat hippocampus via type II glucocorticoid receptors.

We investigated the role of IL-1alpha and IL-1beta expressed in the reactive gliosis following hippocampal damage induced by trimethyltin (TMT). IL-1alpha immunoreactivity was expressed earlier in small glial cells on day 4 post-TMT, while IL-1beta expression was obvious in large swollen glial cells on day 14 post-TMT. Both IL-1alpha and IL-1beta immunoreactivities were double-labeled with astrocyte marker, vimentin, but not with a microglia marker, OX-42. The expression of both IL-1alpha/beta was enhanced by adrenalectomy (ADX) prior to TMT administration. Corticosterone (CORT) or dexamethasone (DEX) supplementation not only cancelled effects of ADX, but also partially reversed TMT-induced enhancement of IL-1alpha/beta expressions. These changes coincided with TMT-induced neuronal death in CA3 pyramidal cells of the hippocampus. It is suggested that IL-1alpha/beta expressed in reactive astrocytes participate in TMT neurotoxicity via type II glucocorticoid receptors.

Voxel-based analysis of MRI reveals anterior cingulate gray-matter volume reduction in posttraumatic stress disorder due to terrorism.

MRI studies using the manual tracing method have shown a smaller-than-normal hippocampal volume in patients with posttraumatic stress disorder (PTSD). However, these studies have yielded inconsistent results, and brain structures other than the hippocampus have not been well investigated. A recently developed, fully automated method called voxel-based morphometry enables an exploration of structural changes throughout the brain by applying statistical parametric mapping to high-resolution MRI. Here we first used this technology in patients with PTSD. Participants were 9 victims of the Tokyo subway sarin attack with PTSD and 16 matched victims of the same traumatic event without PTSD. The voxel-based morphometry showed a significant gray-matter volume reduction in the left anterior cingulate cortex (ACC) in trauma survivors with PTSD compared with those without PTSD. The severity of the disorder was negatively correlated with the gray-matter volume of the left ACC in PTSD subjects. There were no significant differences in other gray-matter regions or any of the white-matter regions between two groups. The present study demonstrates evidence for structural abnormalities of ACC in patients with PTSD. Together with previous functional neuroimaging studies showing a dysfunction of this region, the present findings provide further support for the important role of ACC, which is pivotally involved in attention, emotional regulation, and conditioned fear, in the pathology of PTSD.

Type II glucocorticoid receptors are involved in neuronal death and astrocyte activation induced by trimethyltin in the rat hippocampus.

According to our previous study, trimethyltin (TMT), a neurotoxicant, induces the loss of pyramidal neurons in the rat hippocampus, which is preceded by a transient increase in plasma corticosterone concentration. To address whether this transient activation of the hypothalamopituitary-adrenocortical axis is related to neuronal loss in the hippocampus, we evaluated the effects of bilateral adrenalectomy (ADX) and the chronic supplemental treatment of glucocorticoid receptor agonists after ADX on TMT-induced hippocampal damage. Peroral administration of a single dose of TMT (9 mg/kg body wt) induced the extensive loss of CA3 pyramidal neurons and reactive astrocytosis in the hippocampus, as evidenced by results of vimentin and glial fibrillary acidic protein immunohistochemistry, and the effects were profoundly exacerbated by bilateral adrenalectomy. Prolonged administration of corticosterone not only attenuated the exacerbating effects of adrenalectomy but also partially reversed the TMT-induced neuronal loss and reactive astrocytosis. Dexamethasone, but not aldosterone, could be substituted for corticosterone, suggesting a novel neuroprotective action of type II glucocorticoid receptors in the hippocampus.

Altered hippocampal expression of neuropeptide Y, somatostatin, and glutamate decarboxylase in Ihara's epileptic rats and spontaneously epileptic rats.

By in situ hybridization and immunocytochemistry, expression of neuropeptide Y (NPY), somatostatin and glutamate decarboxylase 65 (GAD65) was studied in the hippocampus of two different epileptic mutant rats, Ihara's epileptic rat (IER) and the spontaneously epileptic rat (SER). GAD65 mRNA expression was enhanced in interneurons of the hippocampus in young IER, that had not yet developed generalized seizures. In older IER and older SER that both showed spontaneous seizures, marked increases of NPY mRNA in hippocampal granule cells and interneurons were found, as well as elevated GAD65 mRNA levels in interneurons. NPY immunoreactivity was enhanced in hilar interneurons and the dentate gyrus of older IER. In addition, some older IER stained heavily for NPY in mossy fibers. These findings suggest that up-regulation of NPY and GAD65 synthesis may be important in epileptogenesis.

Upregulation of brain somatostatin and neuropeptide Y following lidocaine-induced kindling in the rat.

Male Sprague-Dawley rats received a daily injection of 60 mg/kg of lidocaine (> 30 days). Twenty percent of rats developed convulsions (kindled rats) and remaining rats did not show convulsions (non-kindled rats). The level of immunoreactive somatostatin (IR-SRIF) in kindled rats was significantly increased in amygdala than that in non-kindled rats and control rats. Immunoreactive neuropeptide Y (IR-NPY) contents in kindled rats were significantly increased in amygdala, hippocampus, cortex and striatum compared to non-kindled and control rats. The expression of SRIF mRNA in kindled rats produced a significant increase in amygdala, while NPY mRNA in kindled rats showed an elevated expression in both amygdala and hippocampus. These results coincide with the previous findings with the elevated expression of SRIF and NPY mRNA in electrically and pharmacologically kindled models, suggesting the important role of these peptides in the kindling phenomenon.

Paroxysmal kinesigenic choreoathetosis locus maps to chromosome 16p11.2-q12.1.

Paroxysmal kinesigenic choreoathetosis (PKC), the most frequently described type of paroxysmal dyskinesia, is characterized by recurrent, brief attacks of involuntary movements induced by sudden voluntary movements. Some patients with PKC have a history of infantile afebrile convulsions with a favorable outcome. To localize the PKC locus, we performed genomewide linkage analysis on eight Japanese families with autosomal dominant PKC. Two-point linkage analysis provided a maximum LOD score of 10.27 (recombination fraction [theta] =.00; penetrance [p] =.7) at marker D16S3081, and a maximum multipoint LOD score for a subset of markers was calculated to be 11.51 (p = 0.8) at D16S3080. Haplotype analysis defined the disease locus within a region of approximately 12.4 cM between D16S3093 and D16S416. P1-derived artificial chromosome clones containing loci D16S3093 and D16S416 were mapped, by use of FISH, to 16p11.2 and 16q12.1, respectively. Thus, in the eight families studied, the chromosomal localization of the PKC critical region (PKCR) is 16p11.2-q12.1. The PKCR overlaps with a region responsible for "infantile convulsions and paroxysmal choreoathetosis" (MIM 602066), a recently recognized clinical entity with benign infantile convulsions and nonkinesigenic paroxysmal dyskinesias.

Familial paroxysmal kinesigenic choreoathetosis: an electrophysiologic and genotypic analysis.

PURPOSE: We report a pedigree of familial paroxysmal kinesigenic choreoathetosis (PKC) in which five of 18 members are affected. The pathophysiologic basis for PKC is still uncertain; reflex epilepsy versus dysfunction of basal ganglia. We examined (a) whether there were ictal discharges during the attacks, and (b) a linkage between PKC and possible DNA markers linked to several familial epileptic or movement disorders. METHODS: Video-monitoring EEG was performed in two patients with PKC during attacks elicited by movements of the lower extremities. Blood samples for DNA studies were obtained from 15 members of the pedigree. Fourteen polymorphic markers on chromosomes 1p, 2q, 6p, 10q, and 20q were genotyped, and two-point lod scores were calculated for each marker under a dominant model. RESULTS: No ictal discharges were found during the attacks in both patients. We could not obtain significant linkage of PKC with any marker examined. CONCLUSIONS: The video-monitoring EEG findings in our cases strongly suggested that the etiology of PKC should be considered distinct from that of reflex epilepsy. However, the patients in this pedigree had experienced generalized convulsions in their infancies; thus we could not deny the possibility of an epileptogenic basis for PKC. There was no strong evidence for a linkage of the gene for PKC with the candidate regions on 1p, 2q, 6p, 10q, or 20q.

Calcitonin gene-related peptide immunoreactivity in the hippocampus and its relationship to cellular changes following exposure to trimethyltin.

Calcitonin gene-related peptide (CGRP) is a neuropeptide that is regionally regulated following peripheral insult and in central nervous system (CNS) damage models targeting limbic structures. Functional studies have shown this neuropeptide to be involved in neuronal protection and remodeling, vasodilation, immunomodulation, and apoptosis, thus making it an important constituent of the acute phase response. In the present study, we characterized the anatomic expression and distribution of CGRP immunoreactivity (CGRP-IR) after exposure to the toxin, trimethyltin (TMT). We chose this model because TMT causes dramatic changes in the endocrine system, the limbic system, particularly the hippocampus, as well as in the immune response. We have specifically focused on comparing the changes in CGRP-IR with the pattern of apoptosis (via TUNEL staining), cell-cycle activation (Ki67-IR), and in alteration in microglia (OX-42-IR) and astrocyte (gGFAP-IR) immunocytochemistry in TMT-treated hippocampus. Our results show a marked change in CGRP-IR in regions of the hippocampus that are temporally and anatomically correlated with the induction of apoptosis and activation of microglia, astrocyte, and the cell-cycle marker. Given the known effects of CGRP on these cell types and on programmed cell death elsewhere, these findings are consistent with a regional immunoregulatory/injury response role for CGRP following organotin poisoning.

Trimethyltin-induced expression of neuropeptide Y Y2 receptors in rat dentate gyrus.

Trimethyltin (TMT) causes prominent neuronal damage and enhanced expression of neuropeptide Y in the hippocampus. We investigated expression of neuropeptide Y Y2 receptors after TMT intoxication. Markedly elevated (by 470%) concentrations of Y2 receptor mRNA were found in the suprapyramidal blade of the dentate granule cell layer after 5 days. Increases in the infrapyramidal blade were less prominent (by 198%). After 16 days, mRNA levels in both blades of the granule cell layer showed no significant difference from those in controls. Quantification of Y2 receptor-specific binding revealed no significant change at both 5 and 16 days after TMT intoxication. It is suggested, together with a previous report describing a similar increase of neuropeptide Y expression, that a transient expression of Y2 receptors in the dentate gyrus in the initial phase of TMT intoxication may be involved in mediating TMT-induced hippocampal damage.

Evaluation of self-similar features in time series of serum growth hormone and prolactin levels by fractal analysis: effect of delayed sleep and complexity of diurnal variation.

We assayed the diurnal concentrations of growth hormone (GH) and prolactin (PRL) in 6 healthy male volunteers to evaluate the self-similar features in the time series of each hormone on the basis of fractal theory and to determine the fractal dimension as an index of the complexity of the diurnal variation. In addition, we assessed the effects of a 6-hour delay in the sleep period on the complexity of the diurnal variation of these hormones. There was a statistically significant fractal feature in the serum levels of GH both under the nocturnal-sleep and delayed-sleep conditions in all subjects. The time series of the serum PRL concentrations also showed a statistically significant fractal feature under the nocturnal-sleep and delayed-sleep conditions in all subjects. The fractal dimensions of the patterns of the GH or PRL levels were 1.879 and 1.929 or 1.754 and 1.785 under the nocturnal-sleep and delayed-sleep conditions, respectively. Two-way ANOVA revealed no significant difference in the fractal dimension between the two sleep conditions but did reveal a significant difference between the fractal dimensions of the GH and PRL levels. These results showed (1) that delayed sleep had no significant effect on the complexity of the diurnal pattern of these hormones, and (2) that the diurnal pattern of the GH levels was more complex than that of the PRL levels.

Trimethyltin intoxication induces marked changes in neuropeptide expression in the rat hippocampus.

In situ hybridization and immunocytochemistry were applied to investigate changes in the expression of somatostatin, neuropeptide Y, neurokinin B, cholecystokinin, dynorphin, and Met-enkephalin in the rat hippocampus after administration of a single peroral dose of trimethyltin hydroxide (9 mg/kg). Two time intervals were investigated: 5 days after trimethyltin treatment, when CA3 damage becomes manifest and is associated with increased aggression, seizure susceptibility, and memory deficit, and 16 days after trimethyltin, when neuronal damage is almost maximal and seizure susceptibility is declining. Robust but transient increases of neuropeptide Y, neurokinin B, and Met-enkephalin mRNA levels were revealed in the granule cell layer of the dentate gyrus and increased neuropeptide Y and neurokinin B immunoreactivities were found in mossy fibers. In reverse, dynorphin mRNA and immunoreactivity were decreased transiently in the dentate gyrus and mossy fibers, respectively. Strong over-expression of NPY mRNA was also observed in hilar interneurons and in CA1 and CA3 pyramidal cells as well as in the cortex at 5 days postdosing. Cholecystokinin- or neurokinin B-containing basket cells were preserved, while somatostatin-bearing interneurons were damaged by trimethyltin exposure. These neurochemical changes induced by trimethyltin intoxication strikingly parallel to those observed in animal models of temporal lobe epilepsy and may reflect activation of endogenous protective mechanisms. It is also suggested that hilar interneurons respond differently to trimethyltin exposure, for which neuropeptides are valuable markers.

Trimethyltin syndrome as a hippocampal degeneration model: temporal changes and neurochemical features of seizure susceptibility and learning impairment.

The effects of trimethyltin on the hippocampus were investigated in terms of changes in histology, depth electroencephalography, learning acquisition and memory retention, choline acetyltransferase and neuropeptides, and seizure-induced c-fos messenger RNA expression. The results were as follows. (1) Morphologically, trimethyltin produced a progressive loss of hippocampal CA3 and CA4 pyramidal cells, starting from four days after peroral treatment with trimethyltin hydroxide (9 mg/kg), as described previously. (2) Neurophysiologically, the increased seizure susceptibility to pentylenetetrazol treatment reached a maximum at four days post-trimethyltin and then declined after five days post-trimethyltin. The maximal seizure susceptibility at four days post-trimethyltin was confirmed by the immediate and long-lasting appearance of spike discharge in the hippocampus. However, this was not verified by the expression of c-fos messenger RNA in the hippocampus, which was comparable between trimethyltin-treated and control rats. (3) Behaviorally, the time-courses of aggression and learning impairment were similar to that of the seizure susceptibility. (4) Neurochemically, trimethyltin treatment caused changes of neurochemical markers, which were manifested by the elevation of neuropeptide Y content in the entorhinal cortex, and of choline acetyltransferase in the hippocampal CA3 subfield. Trimethyltin may offer potential as a tool for investigations on the relationship between neuronal death in the hippocampus and the development of seizure susceptibility and learning impairment. Alterations in glucocorticoids, glutamate and neuropeptides may all contribute to the manifestation of the trimethyltin syndrome.

Changes of immunoreactive neuropeptide Y, somatostatin and corticotropin-releasing factor (CRF) in the brain of a novel epileptic mutant rat, Ihara's genetically epileptic rat (IGER).

Ihara's genetically epileptic rat (IGER) is a rat mutant with genetically scheduled spontaneous convulsions mimicking human limbic seizures. In the present study, the possible changes of three neuropeptides, neuropeptide Y (NPY), somatostatin (SRIF) and corticotropin-releasing factor (CRF), in the brains of IGER were investigated. Increased contents of immunoreactive (IR) NPY were found only in the hippocampus of 2-month IGERs before developing convulsive seizures, while similar increases of IR-NPY were discovered in the striatum and pyriform and entorhinal cortex as well as hippocampus in 8-month IGERs with repetitive seizures. There were no significant differences in the brain contents of IR-SRIF and IR-CRF between IGERs and the controls at both ages. These findings indicate an enhanced rate of NPY synthesis in this experimental model of epilepsy which may play a critical role in the development of epileptogenesis.

Nitric oxide synthase-containing neurons in the hippocampus are preserved in trimethyltin intoxication.

We studied the effects of trimethyltin (TMT) (9 mg/kg, p.o.) on the nitric oxide synthase (NOS)-containing neurons in the rat hippocampus by NADPH-diaphorase histochemistry and a biochemical assay of NOS activity. TMT exposure caused the typical behavioral changes and a loss of the CA3/4 pyramidal cells, which were NADPH diaphorase-negative. The scattered interneurons and the CA1 pyramidal cells, which were NADPH diaphorase-positive, were spared. Hippocampal NOS activity showed no reduction in the TMT-treated rats compared with the controls. These results provide evidence of the preservation of the NOS-containing neurons in TMT intoxication.

The role of vasopressin, somatostatin and GABA in febrile convulsion in rat pups.

In order to further elucidate a possible role of neuropeptides and GABA in the pathogenesis of febrile convulsions, we studied changes of immunoreactive-arginine vasopressin (IR-AVP), IR-somatostatin (IR-SRIF) and gamma-aminobutyric acid (GABA) in the rat brain after febrile convulsions induced by ultra-red light (UR). Male Wistar rats at 16 days of age irradiated with UR developed generalized convulsions after 4.9 +/- 0.5 min irradiation. Six rats were killed by microwave irradiation 3 min after UR irradiation prior to convulsion development, and 29 rats were killed either 0 min, 2 h, 6 h, 24 h or 48 h after febrile convulsions. Non-irradiated rats served as controls. The rat brain was dissected into 4 regions; amygdala, hypothalamus, cortex and hippocampus, and subjected to radioimmunoassays. IR-AVP levels in hypothalamus were increased 3 min after UR and decreased at 2 h and 6 h after the convulsions. IR-SRIF levels were increased in cortex and hippocampus at 3 min after UR and 0 min after the convulsions. The GABA content increased in all regions tested at 2 h and 6 h after the convulsions. These results suggest that AVP, SRIF and GABA may be involved in the pathogenesis of febrile convulsions in different ways.

The 24-hour rhythms in plasma growth hormone, prolactin and thyroid stimulating hormone: effect of sleep deprivation.

Twenty-four hour secretory rhythms of growth hormone (GH), prolactin (PRL) and thyroid stimulating hormone (TSH) were investigated in 9 normal adult men by means of serial blood sampling at 30 min intervals. The profiles of pituitary hormones were compared in 6 subjects between in normal nocturnal sleep condition and in delayed sleep condition. Plasma GH was measured with use of highly sensitive enzyme immunoassay (EIA) recently developed. Plasma TSH was also evaluated by highly sensitive time-resolved fluorometric immunoassay (TR-FIA). Time series analysis of plasma GH and PRL was performed by auto- and cross- correlation and spectral analysis. The detection limit of EIA for GH was 0.3 pg/ml and all plasma GH levels were within the detectable range of this EIA. Cross-correlation and spectral analysis suggested the presence of approximately 2-3 h rhythmicity of plasma GH. Plasma PRL appeared to have some 24-hour rhythmicity besides its sleep-dependent component. Sleep deprivation caused marked elevation of plasma TSH during night time. It is suggested that there appears two mechanisms regulating GH secretion: one has a sleep-independent and ultradian rhythm and another has a sleep-dependent rhythm.

Altered brain contents of neuropeptides in spontaneously epileptic rats (SER) and tremor rats with absence seizures.

Immunoreactive- (IR-) somatostatin (SRIF), neuropeptide Y (NPY) and corticotropin-releasing factor (CRF) contents were investigated in the brain of tremor rats with absence-like seizure and spontaneously epileptic rats (SER), which is a genetically defined double-mutant (zi/zi, tm/tm) obtained by mating zitter homozygote (zi/zi) with tremor heterozygote (tm/+) and shows both absence-like seizure and tonic convulsions. Increased levels of IR-NPY and IR-CRF were observed in several regions including the amygdala and hippocampus in homozygous SER compared to heterozygous SER (zi/zi, tm/+ or +/+). Homozygous tremor rats (tm/tm) showed lower levels of IR-NPY and IR-CRF contents mainly in the hippocampus and mesolimbic system (entorhinal and pyriform cortex and nucleus accumbens) than heterozygous tremor rats. IR-SRIF contents of homozygous SER were higher in frontal cortex than heterozygous SER and in amygdala than homozygous tremor rats. No change of IR-SRIF between groups was noted in the hippocampus among brain structures underlying epileptogenicity. The results suggest that the change of neuropeptide levels, most conspicuous in NPY among three peptides tested, may be involved in the phenotypical manifestation of seizures in SER and tremor rats, and that the development of tonic convulsion and absence seizures may be differently associated with the change of brain neuropeptide levels.