Neonatal rotenone lesions cause onset of hyperactivity during juvenile and adulthood in the rat.

Attention deficit hyperactivity disorder (ADHD) is characterized by behavioral and cognitive symptoms. Longitudinal studies demonstrated that the symptoms remains clinically significant for the majority of ADHD children into adulthood. Furthermore, a population-based birth cohort provided the initial evidence of adult ADHD that lacks a history of childhood ADHD. We previously demonstrated that neonatal exposure to bisphenol A, an environmental chemical caused hyperactivity in the juvenile. Here, we extend to examine other chemical such as rotenone, a dopaminergic toxins. Oral administration of rotenone (3mg/kg) into 5-day-old male Wistar rats significantly caused hyperactivity at adulthood (8∼11 weeks old; p<0.05). It was about 1.3∼1.4-fold more active in the nocturnal phase after administration of rotenone than control rats. Higher dose (16mg/kg) or repeated lower dose of rotenone (1mg/kg/day for 4days) caused hyperactivity in the juvenile. Furthermore, DNA array analyses showed that neonatal exposure to rotenone altered the levels of gene expression of several molecules related to apoptosis/cell cycle, ATPase, skeletal molecule, and glioma. Bivariate normal distribution analysis indicates no correlation in gene expression between a hyperactivity disorder model and a Parkinson's disease model by rotenone. Thus, we demonstrate a rotenone models of ADHD whose onset varies during juvenile and adulthood.

Activation of STAT3 by pituitary adenylate cyclase-activating polypeptide (PACAP) during PACAP-promoted neurite outgrowth of PC12 cells.

Addition of pituitary adenylate cyclase-activating polypeptide (PACAP) into the cultured PC12 cells promoted neurite outgrowth of the cells, indicating cell differentiation. Using DNA macroarray techniques, we have characterized the PC12 cell transcriptome, revealing that several genes were regulated by PACAP. Among many, we focused to investigate whether STAT3 molecule, whose message was up-regulated, might be involved in PACAP signaling. Reverse transcription polymerase chain reaction supported the results obtained by DNA macroarray; PACAP increased gene expression of STAT3, at least up to 24 h, being a maximum 9.5-fold with 3 h-treatment of 1 nM PACAP. Reporter assay analyses demonstrated that PACAP activated STAT3-response promoter, but neither GAS- nor ISRE-response promoters. Enzyme-linked immunosorbent assay revealed that PACAP increased the amount of STAT3 proteins about 30%. Concurrently, phospho-STAT3 Tyr705 was increased in the nuclei by the neuropeptide. Ectopic expression of dominant negative forms of STAT3, which had Tyr705 to phenylalanine substitution, effectively decreased PACAP-promoted neurite extension (55%). During the activation of STAT3, the secretion of interleukin-6 (IL-6) was stimulated by PACAP in a dose-dependent manner. Treatment with IL-6-neutralizing antibody significantly diminished PACAP-activated STAT3 promoter activity and PACAP-induced neurite outgrowth. Thus, our findings have demonstrated that STAT3 is involved in PACAP signaling and function during PC12 cell differentiation by the neuropeptide.

Behavioural characteristics and gene expression in the hyperactive wiggling (Wig) rat.

Recently, congenic wiggling (Wig) rats were described as a good model for attention-deficit hyperactivity disorder; 12- to 14-week-old animals demonstrated hyperactivity, impulsive behaviour and an impaired working memory. Here, we show that 4- to 5-week-old Wig rats displayed significantly greater spontaneous motor activity than control rats during a period of darkness. Subcutaneous injection of 4 mg/kg methamphetamine exacerbated hyperactivity, the reverse of its effect in rats with neonatally induced 6-hydroxydopamine lesions. Immunohistochemistry showed low levels of tyrosine hydroxylase in the ventral midbrain, similar to 6-hydroxydopamine-treated rats. In cDNA macroarrays, 4-week-old Wig rats showed increased expression of the adenosine A2a receptor in the dorsal striatum, macrophage migration inhibitory factor in the frontal cortex, ventral striatum and midbrain, and calbindin 2 in the dorsal and ventral midbrain. Expression of the gamma-aminobutyric acid (GABA) transporter and sterol carrier protein 2 genes was reduced in all regions. Dopamine transporter gene expression was increased in the dorsal midbrain but decreased in the ventral midbrain, a pattern distinct from that induced by 6-hydroxydopamine. Although abnormal development of dopaminergic neurons may underlie motor hyperactivity, other mechanisms may control responsiveness to methamphetamine. Wig rats may provide a model of attention-deficit hyperactivity disorder in which treatment with psychostimulants accelerate the hyperactivity.

Melatonin inhibits maneb-induced aggregation of alpha-synuclein in rat pheochromocytoma cells.

Melatonin, a secretory product of the pineal gland, is involved in the regulation of circadian and seasonal rhythms, in oncostasis, and in inducing osteoblast differentiation. Furthermore, melatonin is a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. In this study, the antioxidant nature of melatonin was shown to prevent cultured neural cells from apoptosis induced by endocrine-disrupting chemical, maneb. The neurotoxicity of the fungicide, maneb (1 microg/mL), on the PC12 cells was elicited through apoptotic cell death, concomitant with aggregation of alpha-synuclein, a feature of Parkinson's disease. Activation of caspase-3/7 was associated with this process. A fluorescence rationing technique using a mitochondrial dye revealed that maneb altered the mitochondrial membrane potential of the neural cells. However, melatonin (1 nm) largely prevented the neural cells from the neural toxicant by inhibition of both caspase-3/7 activation and disruption of the mitochondrial transmembrane potential. Furthermore, aggregation of alpha-synuclein by maneb was also inhibited by melatonin. Thus, melatonin prevents maneb-induced neurodegeneration at a nighttime physiological blood concentration, most likely by inhibiting the aggregation of alpha-synuclein as well as preventing mitochondrial dysfunction in PC 12 cells.

Region-dependent differences and alterations of protective thiol compound levels in cultured astrocytes and brain tissues.

We examined region-dependent differences and alterations in the levels of protective thiol compounds, glutathione (GSH) and metallothionein (MT)-I and -II, in cultured rat astrocytes under several culture conditions and in brain tissues of rats at postnatal and weaning periods. Regardless of culture conditions, both protein concentrations and mRNA expressions of MT-I and -II were much higher in the cerebral hemisphere than in cerebellar astrocytes, whereas no difference was observed in GSH concentration. In both astrocytes, the GSH concentrations did not change within 12 h but significantly increased 24 h after being maintained in a serum-free defined medium. At 24 h, protein concentrations and mRNA expressions of MT-I and -II also increased in the respective astrocytes, and were further enhanced when maintained in the presence of 50 microM Zn(2+). In the brain tissues, the MT-I/-II protein concentrations were significantly higher in the cerebral cortex (a part of the cerebral hemisphere) than in the cerebellum, whereas the GSH concentration was similar at both postnatal day (P)1 and P35. In addition, the concentrations in the respective regions were significantly higher at P35 than at P1. These results suggest that region-dependent differences in the cellular levels of GSH and MTs in cultured astrocytes might reflect the in vivo differences, and that the levels of the respective thiol compounds in cultured astrocytes increase after serum elimination along with the region-dependent differences.

Overexpression of Bcl-2 inhibits nuclear localization of annexin I during tumor necrosis factor-alpha-mediated apoptosis in porcine renal LLC-PK1 cells.

The addition of tumor necrosis factor (TNF)-alpha into the cultured porcine kidney LLC-PK1 cells caused apoptosis concomitantly with caspase-3 activation and the inductions of an endogenous Bcl-2 protein. An SDS-polyacrylamide electrophoretic analysis revealed that a 37-kDa protein in a nuclear fraction was increased during TNF-alpha-induced apoptosis. Partial amino acid sequence of the protein was A-L-T-G-H-L-E-E-V, perfectly matching that of annexin I. Immunocytochemistry revealed that annexin I migrated to the nucleus and/or peri-nucleus region upon exposure to TNF-alpha. Overexpression of Bcl-2 proteins inhibited the nuclear localization of annexin I during TNF-alpha-induced apoptosis. Antisense oligodeoxynucleotides complementary to annexin I-inhibited TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labelling) staining in TNF-alpha-treated cells, suggesting that annexin I expression is a possible prerequisite for the induction of apoptosis by the cytokine. Thus, it is first time to show that annexin I is regulated by an anti-apoptotic Bcl-2 protein in TNF-alpha-induced renal apoptotic events.

Motor hyperactivity caused by a deficit in dopaminergic neurons and the effects of endocrine disruptors: a study inspired by the physiological roles of PACAP in the brain.

Recent studies have revealed that the pituitary adenylate cyclase-activating polypeptide (PACAP) might act as a psychostimulant. Here we investigated the mechanisms underlying motor hyperactivity in patients with pervasive developmental disorders, such as autism, and attention-deficit hyperactivity disorder (ADHD). We studied the effects of intracisternal administration of 6-hydroxydopamine (6-OHDA) or endocrine disruptors (EDs) on spontaneous motor activity (SMA) and multiple gene expression in neonatal rats. Treatment with 6-OHDA caused significant hyperactivity during the dark phase in rats aged 4-5 weeks. Motor hyperactivities also were observed after treatment with endocrine disruptors, such as bisphenol A, nonylphenol, diethylhexyl phthalate and dibutyl phthalate, during both dark and light phases. Gene-expression profiles produced using cDNA macroarrays of 8-week-old rats with 6-OHDA lesions revealed the altered expression of several classes of gene, including the N-methyl-D-aspartate (NMDA) receptor 1, glutamate/aspartate transporter, gamma-aminobutyric-acid transporter, dopamine transporter 1, D4 receptor, and peptidergic elements such as the galanin receptor, arginine vasopressin receptor, neuropeptide Y and tachykinin 2. The changes in gene expression caused by treatment with endocrine disruptors differed from those induced by 6-OHDA. These results suggest that the mechanisms underlying the induction of motor hyperactivity and/or compensatory changes in young adult rats might differ between 6-OHDA and endocrine disruptors.

Transcriptome of pituitary adenylate cyclase-activating polypeptide-differentiated PC12 cells.

Addition of pituitary adenylate cyclase-activating polypeptide (PACAP) into the cultured PC12 cells secreted dopamine and promoted neurite outgrowth of the cells, indicating cell differentiation. To characterize the PACAP-differentiated PC12 cell transcriptome, we applied DNA macroarray techniques, using Atlas Rat 1.2 Array membranes (BD Biosciences Clontech) that have 1176 cDNA. RNA samples were harvested from PC12 cells before and at a time of 6 h treatment with 1 nM PACAP, when neuritogenesis was remarkably observed under the condition used. Several genes regulated by PACAP have been associated with neuritogenesis (i.e. villin 2 and tissue plasminogen activator) or cell growth/differentiation (i.e. cyclin or ornitine decarboxylase). Also, cytoskeleton proteins such as actin or tubulin were up-regulated for cell morphology remodeling. A message of vehicle trafficking molecule (synaptotagmin IV) was more remarkably increased (3.95-6.85-fold). Signaling molecules such as small G proteins (rab12, rab16, or ral), IkappaB, or STAT3 were altered by PACAP. It is noteworthy that PACAP inhibited the expression of galanin receptor 2, whose ligand was shown to inhibit tyrosine hydroxylase activity. Thus, in this study the transcriptome of PACAP-differentiated PC12 was established, leading to the elucidation of the molecular mechanism of neuritogenesis by the neuropeptide.

p-Nitrotoluene causes hyperactivity in the rat.

It has not been known which endocrine disruptors exert their effects on neuronal functions, particularly leading to behavioral alterations. To address this, we examined the effects of p-nitrotoluene, an endocrine disruptor, on rat behavior and gene expression. Single intracisternal administration of p-nitrotoluene (ca. 10 microg) into 5-day-old male Wistar rats caused significant hyperactivity at 4-5 weeks of age. They were about 1.4-fold more active in the nocturnal phase after administration of p-nitrotoluene than control rats. Based on DNA array analyses, p-nitrotoluene decreased more than two-fold the levels of gene expression of the mesencephalic dopamine transporter at 8 weeks old. Thus, it was demonstrated for the first time that p-nitrotoluene definitely affected the developing brain, resulting in hyperactivity in the rat.

Transient inhibition of synergistically insulin-like growth factor-1- and bisphenol A-induced poliferation of estrogen receptor alpha (ERalpha)-positive human breast cancer MCF-7 cells by melatonin.

There are many subclones of human breast cancer MCF-7 cells that respond to different levels of estrogen and that have been used for evaluating the estrogenic potential of environmental chemicals such as bisphenol A. In this study, I examined the effects of melatonin, an endogenous growth-inhibitory hormone, on the bisphenol A-induced proliferation of an MCF-7 subclone, designated as MCF-7-EMF cells. MCF-7-EMF cell growth was extremely slow in the presence of either estrogen at 10(-7) M or bisphenol A at 10(-7) M in a phenol-red-free medium and 10% charcoal-stripped fetal bovine serum. It was difficult to detect the increment of BrdU incorporation in MCF-7-EMF cells (2x10(4) cells) upon exposure to 10(-7) M bisphenol A alone for up to 50 h. The same result was obtained with cells exposed to 10 ng/ml insulin-like growth factor-1 (IGF-1) alone for up to 50 h. However, in the presence of 10 ng/ml IGF-1, bisphenol A markedly increased cell proliferation. The synergistic response was transiently inhibited by 10(-10) M melatonin (p<0.05). Thus, melatonin caused a transient inhibition of cell growth of estrogen receptor alpha (ERalpha)-positive MCF-7-EMF cells induced synergistically by both IGF-1 and bisphenol A.

Influence of cell density and thyroid hormone on glial cell development in primary cultures of embryonic rat cerebral hemisphere.

The influence of cell density and thyroid hormone (TH) on the development of astrocytes and oligodendrocytes was investigated in primary cultures prepared from rat cerebral hemisphere on embryonic day (E)18. At the beginning of the culture, most of the cells were microtubule-associated protein 2 (MAP2)-positive neurons, whereas O1-positive oligodendrocytes and glial fibrillary acidic protein (GFAP)-positive astrocytes were rarely observed. After the cells were maintained in serum-free defined medium, astrocytes developed at high cell density but rarely at a low one. When leukemia inhibitory factor (LIF) was supplemented in low-density cultures, the levels of GFAP expression markedly increased to almost the same extent as in high-density culture without TH. This suggests that, in low-density cultures, astrocyte progenitors could not differentiate because of insufficient astrocyte-inducing factors. Interestingly, the addition of TH increased GFAP expression levels only at high density. The number of oligodendrocytes increased with TH addition at both cell densities, although the effects were more remarkable at high density. These results suggest that cell density and TH are pivotal factors in the development of both astrocytes and oligodendrocytes. It is also suggested that the effects of TH on glial cell development could be accelerated via cell-cell communications.

Attenuation of both apoptotic and necrotic actions of cadmium by Bcl-2.

We examined the effects of cadmium on the bcl-2 family of proteins--bcl-2, bax, bad, and bcl-xS/L--in cadmium-induced cytotoxicity. Addition of 10 microM cadmium to cultured porcine kidney LLC-PK(1) cells caused apoptosis. Western blot analyses revealed that cadmium markedly increased endogenous bcl-2 protein (to 3-4 times the level in wild-type cells) earlier than metallothionein induction, but that the metal did not enhance the induction of bax, bad, or bcl-xS proteins. Cadmium also induced the transcript of bcl-2, with the amount of bcl-2 reaching a maximum at 1-2 hr of exposure; this increase occurred earlier than cadmium-induced increase in the protooncogene such as c-myc. A cadmium-induced increase in endogenous bcl-2 protein was also seen in rat primary thymocytes. Overexpression of the bcl-2 protein by gene transfection prevented cadmium-induced apoptosis. Following the detection of apoptosis, lactate dehydrogenase release in the culture medium (a marker of necrosis) was observed, and this release was also inhibited by overexpression of bcl-2. Electron microscopic observations also supported the fact that cadmium induced apoptotic chromatin condensation at an early stage of exposure, followed by necrotic features of the cells, both of which were also inhibited by overexpression of bcl-2 proteins. Thus, our data demonstrated that both apoptotic and necrotic actions of cadmium were attenuated by bcl-2.