Egr1 involvement in evening gene regulation by melatonin.

Seasonal photoperiodic responses in mammals depend on the pineal hormone melatonin. The pars tuberalis (PT) region of the anterior pituitary has emerged as a principal melatonin target tissue, controlling endocrine responses. Rising melatonin levels acutely influence the expression of a small cluster of genes either positively (exemplified by cryptochrome-1, cry1) or negatively (exemplified by the type 1 melatonin receptor, mt1). The purpose of this study was to characterize the pathways through which these evening actions of melatonin are mediated. In vitro experiments showed that cAMP signaling in the PT directly influences mt1 but not cry1 expression. Analysis of nuclear extracts from sheep PT tissue collected 90 min after melatonin or saline control injections highlighted the response element for the immediate early gene egr1 (EGR1-RE) as a candidate for acute melatonin-dependent transcriptional regulation. We identified putative EGR1-RE's in the proximal promoter regions of the ovine cry1 and mt1 genes, and confirmed their functionality in luciferase reporter assays. Egr1 expression is suppressed by melatonin in PT cell cultures, and is rhythmic in the ovine PT with a nadir in the early night. We propose that melatonin-dependent effects on EGR1-RE's contribute to evening gene expression profiles in this pituitary melatonin target tissue.

Decreased maternal behavior and anxiety in ephrin-A5-/- mice.

During development of the nervous system, molecular signals mediating cell-cell interactions play critical roles in the guidance of axonal growth and establishment of synaptic functions. The Eph family of tyrosine kinase receptors and their ephrin ligands has been shown to mediate neuronal interactions in the development of topographic axon projection maps in several brain regions, and the loss of Eph activities result in defects in select axonal pathways. However, effects of deficiencies of the Eph signals on animal behavior have not been well documented. In this study, we showed that inactivation of a ligand of the Eph receptors, ephrin-A5, resulted in defects in maternal behavior and alterations in anxiety. Female ephrin-A5 -/- mice show significant defects in nest building and pup retrieval. In addition, lower levels of anxiety were observed in both male and female null mice. These changes were not due to deficiencies in estradiol, progesterone or corticosterone levels. Our observations suggest that ephrin-A5 plays a key role in the development and/or function of neural pathways mediating mouse maternal care and anxiety.

Trimethyltin-induced alterations in behavior are linked to changes in PSA-NCAM expression.

The neurotoxic heavy metal trimethyltin (TMT) primarily damages neurons of the hippocampus and limbic areas of the temporal lobe, and causes a dose-dependent decrease in the polysialated form of the neural cell adhesion molecule (PSA-NCAM) in the mouse hippocampus. In the current study, we attempted to associate deficits in spatial learning following TMT exposure at various stages in learning with changes in levels of NCAM-180 and PSA-NCAM in both the hippocampus and frontal cortex. Mice were treated with TMT either before or after training on a spatial learning paradigm and examined for changes in NCAM and PSA-NCAM 12h later. In the first set of experiments, male BALB/c mice were injected with TMT (2.25 mg/kg) or saline i.p. and tested 24-168 h later using hidden and visible versions of the water maze, as well as light avoidance and motor activity. Mice in both treated and control groups which demonstrated a significant improvement in water maze performance also showed an elevation in hippocampal PSA-NCAM at all time points examined. TMT exposure impaired spatial learning and blocked learning-induced elevations in PSA-NCAM expression 24-96 h post-treatment, but these deficits disappeared by 168 h post-treatment. Mice exposed to TMT during reconsolidation of spatial learning (after repeated water maze training) demonstrated a mild and transient difference in escape latency compared to saline exposed mice. TMT administration during this period did not result in the attenuation of PSA-NCAM expression observed when animals were exposed before training. These results confirm a specific role for PSA-NCAM in acquisition and consolidation of spatial memory.

Spectroscopic properties of ferrous heme complexes of sterically hindered ligands.

Mesoheme IX complexes of sterically hindered ligands 2-methylimidazole, tert-butylamine and 2-methylpyridine in aqueous glycerol solutions are characterized by broad visible absorption spectra at ambient temperature exhibiting close similarities to high-spin ferrous hemeproteins. Spectrophotometric titrations of mesoheme IX with these ligands indicate well-defined equilibria for 2-methylimidazole and tert-butylamine corresponding to the formation of penta-coordinate strong-field ligand complexes. Variable temperature spectra of these complexes from ambient to 77 degrees K exhibit a change to hemochrome spectra characteristic of the low-spin unhindered ligand complexes. Corresponding changes in the visible spectra are not observed for the high-spin hemeproteins deoxymyoglobin, horse-radish peroxidase and cytochrome c. The appropriate utilization of these hindered ligand heme complexes as model systems for high-spin ferrous hemeproteins has been discussed.

The effect of temperature on the spectroscopic properties of the heme c octapeptide from cytochrome c.

The effect of temperature on the optical properties of the acetylated heme c octapeptide from cytochrome c was examined. At above ambient temperatures the observed optical spectrum with maxima at 549 and 424 nm was characteristic of high-spin ferrous hemeproteins. At below ambient temperatures the optical spectrum became characteristic of low-spin ferrous hemeproteins with maxima at 547, 518, and 410 nm. A thermodynamic characterization of this two component system yielded a deltaHO of -10.1 +/- 0.7 kcal/mol and a delta S0 of-37.6 +/- 2.5 e.u. for the temperature dependent process. Discussion of the spectroscopic and thermodynamic parameters was presented in terms of the consistent magnetic and structural properties of heme complexes.

High-field, variable-temperature Mössbauer effect measurements on oxyhemeproteins.

We measured Mössbauer spectra of human oxyhemoglobin, its isolated beta chains, and of oxymyoglobin from horse and sperm whale in fields of 4 or 6 T between 4.2 and 200 K in order to characterize the electronic state of the oxyheme complex. Diamagnetic sodium nitroprusside measured under the same conditions served as a control. The spectra of all compounds are reproduced adequately by a model that assumes a diagmagnetic iron and treats the quadrupole splitting, the asymmetry parameter and the Mössbauer linewidth as adjustable parameters. The results provide no indication in the oxyhemeproteins of the excited triplet state that was postulated by Cerdonio and co-workers (Cerdonio, M., Congiu-Castellano, A., Mogno, F., Pispisa, B., Romani, G.L. and Vitale, S. (1977) Proc. Natl. Acad. Sci. USA 74, 398-400) on the basis of susceptibility measurements on oxyhemoglobin.

Increased excretion of a lipid peroxidation biomarker in autism.

It is thought that autism could result from an interaction between genetic and environmental factors with oxidative stress as a potential mechanism linking the two. One genetic factor may be altered oxidative-reductive capacity. This study tested the hypothesis that children with autism have increased oxidative stress. We evaluated children with autism for the presence of two oxidative stress biomarkers. Urinary excretion of 8-hydroxy-2-deoxyguanosine (8-OHdG) and 8-isoprostane-F2alpha (8-iso-PGF2alpha) were determined in 33 children with autism and 29 healthy controls. 8-iso-PGF2alpha levels were significantly higher in children with autism. The isoprostane levels in autistic subjects were variable with a bimodal distribution. The majority of autistic subjects showed a moderate increase in isoprostane levels while a smaller group of autistic children showed dramatic increases in their isoprostane levels. There was a trend of an increase in 8-OHdG levels in children with autism but it did not reach statistical significance. There was no significant correlation between the levels of the biomarkers and vitamin intake, dietary supplements, medicine, medical disorders, or history of regression. These results suggest that the lipid peroxidation biomarker is increased in this cohort of autistic children, especially in the subgroup of autistic children.

Absorption spectra of cytochrome P450CAM in the reaction with peroxy acids.

The reaction of Fe(III) cytochrome P450CAM with m-chloroperbenzoic acid was studied by rapid scanning absorption spectroscopy. Native low-spin enzyme produced spectra characteristic of two reaction phases that were marked by time intervals with isosbestic positions. The high-spin enzyme substrate complex yielded a series of Soret-region spectra whose properties were dependent on peracid concentration. The simplest model describing the results was a sequence of at least two spectral intermediates, that were not entirely homologous with data measured in reactions with microsomal P450LM2. Comparisons with related heme protein states indicate higher Fe(IV) oxidation levels provide a plausible interpretation of the P450CAM spectra.

Amphetamine induces depletion of dopamine and loss of dopamine uptake sites in caudate.

Long-lasting depletion of dopamine and concomitant loss of dopamine uptake sites follow repeated administration of methylamphetamine to rats. We found similar effects after similar treatment with d-amphetamine, but not after treatment with methylphenidate. Methylphenidate also failed to produce long-term depletion of regional catecholamine levels in rhesus monkeys. These long-lasting alterations of the dopaminergic system suggest that amphetamines or their metabolites have toxic interactions with dopaminergic neurons, which do not occur with methylphenidate.

Age-dependent effects of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP).

The neurotoxic effects of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) have been shown to be dependent upon its conversion to 1-methyl-4-phenyl pyridine ion (MPP+) by monoamine oxidase. Since central monoamine oxidase activity increases with age, the neurotoxic effects of MPTP should be age-dependent. This hypothesis was evaluated by administering MPTP to three groups of rats differing in age. It was observed that both the lethality and neurotoxicity of MPTP were age-dependent.

Methamphetamine-induced neuronal damage: a possible role for free radicals.

The hypothesis that methamphetamine-induced neuronal damage is mediated by the production of free radicals was evaluated by pretreating rats with either antioxidants or a superoxide dismutase (SOD) inhibitor. It was found that methamphetamine (dose range 6.25-25.0 mg/kg) caused long-lasting depletions of dopamine and serotonin in the striatum and that pretreatment with the antioxidants, ascorbic acid (10-100 mg/kg), ethanol (1 g/kg), mannitol (2 g/kg), or vitamin E (2 g/kg), attenuated these depletions, whereas pretreatment with the superoxide dismutase inhibitor diethyldithiocarbamate (200-400 mg/kg) exacerbated the depletions. The alteration of this effect by four different antioxidants, as well as an inhibitor of superoxidase dismutase, indicated that oxygen-free radicals may have a role in the methamphetamine-induced neurotoxicity.

Partial 6-hydroxydopamine-induced lesions and haloperidol-induced catalepsy.

Rats with partial (36%) 6-hydroxydopamine-induced lesions of the striatal dopamine system or sham-lesioned controls were tested for duration of catalepsy after 1.0, 2.0 and 4.0 mg/kg haloperidol. Thereafter, half of each group was given haloperidol (2.0 mg/kg) chronically for 84 days. The cataleptic responses to the 3 doses were tested again on days 13-15 and 74-76 of the chronic injections. Animals that were lesioned and treated with haloperidol chronically had longer durations of catalepsy at the first two dose-response determinations. At the third dose-response determination, there were no differences among the 4 groups. Additionally, all groups displayed an increase in duration of catalepsy at the second and third dose-response determinations, relative to the first. The increase in haloperidol-induced duration of catalepsy in the lesioned group suggests that lower levels of dopamine in the striatum may potentiate extrapyramidal side-effects.

Ascorbic acid reduces the dopamine depletion induced by methamphetamine and the 1-methyl-4-phenyl pyridinium ion.

The neurotoxic actions of methamphetamine and the 1-methyl-4-phenyl pyridinium ion (MPP+) have been ascribed, at least in part, to the generation of free radicals. This hypothesis was evaluated by attempting to reduce the toxic actions of these compounds by pretreatment with an antioxidant, ascorbic acid. The intrastriatal administration of methamphetamine caused a 37% depletion of dopamine. Treatment with 100 or 1000 mg/kg of ascorbic acid significantly reduced the methamphetamine-induced dopamine depletion (by 21 and 27%, respectively). The intrastriatal administration of MPP+ caused an 88% depletion of dopamine. Treatment with 100 or 1000 mg/kg of ascorbic acid significantly reduced the MPP+-induced dopamine depletion (by 22 and 45%, respectively). Thus, free radicals may mediate the toxic actions of these compounds.

Ascorbic acid reduces the dopamine depletion induced by MPTP.

The administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice was found to cause a long-lasting depletion of striatal dopamine concentrations, but did not alter striatal serotonin concentrations. The concomitant administration of ascorbic acid attenuated this MPTP-induced dopamine depletion. These observations are discussed in reference to the possible mechanisms through which MPTP exerts its neurotoxic actions on dopaminergic neurons.

Methamphetamine-induced neurotoxicity in BALB/c, DBA/2N and C57BL/6N mice.

Repeated administration of methamphetamine (METH; 2 and 4 mg/kg, s.c. four times every 2 h) caused hyperthermia and a dose-dependent depletion of striatal dopamine levels 3 days after the METH-treatment in both BALB/cAnNCrj (BALB) and DBA/2NCrj (DBA) mice, but these responses were lower in C57BL/6NCrj (C57BL) mice. An acute decrease of striatal dopamine levels 30 min after the last injection of METH (4 mg/kg) was observed in both BALB and DBA mice, while an increase in dopamine was observed in C57BL mice. Striatal 3-methoxytyramine levels were drastically increased in both DBA and C57BL mice after this same treatment. Moreover, pretreatment with the superoxide dismutase inhibitor, diethyldithiocarbamate (200 mg/kg, i.p.) exacerbated the METH (4 mg/kg)-induced striatal dopamine-depletion in BALB mice. In addition, pretreatment with an inhibitor of poly(ADP-ribose) polymerase, benzamide (160 mg/kg, s.c.), significantly attenuated the METH (4 mg/kg)-induced striatal dopamine depletion in both BALB and DBA mice. These results suggest that both BALB and DBA mice possess a higher sensitivity to the METH-induced striatal dopaminergic neurotoxicity compared to C57BL mice. In addition, the striatal dopaminergic neurons of BALB mice may be more vulnerable to METH-induced oxidative stress as compared to that in C57BL mice.

Methamphetamine-induced striatal dopamine neurotoxicity and cyclooxygenase-2 protein expression in BALB/c mice.

The expression of cyclooxygenase-2 (COX-2) and striatal dopamine (DA) depletion in BALB/cAnNcrj (BALB/c) mice following a neurotoxic dose of methamphetamine (METH) was investigated. METH-treatment (4 mg/kg x 4, 2 h intervals, s.c.) induced a significant hyperthermia and a persistent depletion of striatal DA levels 72 h after the treatment. COX-2, a marker of the cytotoxic effect of inflammation and oxidative stress and thiobarbituric acid (TBA) were significantly induced in the striatum 72 h after the METH-treatment, but not in the hippocampus. These results suggest that COX-2 may participate in METH-induced neurotoxicity in striatum.

Effect of gestational and lactational 2,3,7, 8-tetrachlorodibenzo-p-dioxin exposure on the level and catalytic activities of hepatic microsomal CYP1A in prepubertal and adult rats.

We determined the inducibility, as well as the persistence of the induction, of hepatic microsomal CYP1A1 and CYP1A2 (by western blot analysis), and their catalytic activities (as measured by resorufin ether O-dealkylation) in prepubertal (25-day-old) and adult (120-day-old) offspring of timed-pregnant Sprague-Dawley rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD treatment was subcutaneous, at a low dose of 0.1 microg/kg, on gestational days 7, 14, and 20, and on lactational days 7 and 14. CYP1A1 protein was induced significantly (23-fold) in prepubertal but not in adult offspring of TCDD-exposed dams, whereas ethoxyresorufin O-deethylase (EROD) activity, which is CYP1A1-preferential, was induced less extensively (5-fold) and slightly (1.7-fold) in the prepubertal and adult offspring, respectively. Benzyloxyresorufin O-debenzylase (BROD) activity, which is CYP2B-preferential but has been reported to be catalyzed by CYP1A1, was also induced 5- and 6-fold in prepubertal and adult offspring, respectively, of TCDD-exposed dams. However, the induced BROD activity was neither inhibited by antibody against CYP1A1 nor accompanied by an elevated level of microsomal CYP2B. CYP1A2 was induced slightly only in prepubertal offspring of TCDD-treated dams. There was suggestive evidence of enhanced lipid peroxidation in hepatic microsomes from prepubertal but not adult offspring of TCDD-treated dams. These data showed that in utero plus lactational TCDD exposure effected transient induction of hepatic microsomal CYP1A1 but sustained induction of BROD activity, which may be catalyzed by enzymes other than CYP1A or CYP2B.

Neurochemical and behavioral deficits consequent to expression of a dominant negative EphA5 receptor.

The Eph family tyrosine kinase receptors and their ligands have been linked to axon guidance and topographic mapping of the developing central nervous system. More specifically, the EphA5 receptor has been shown to play a role in development of hippocamposeptal, retinotectal and thalamocortical projections. Recently, a line of transgenic mice was developed which expresses a truncated EphA5 receptor lacking a functional tyrosine kinase domain. In a previous study, axonal tracing revealed that medial hippocampal axons in this strain projected laterally and ventrally away from their normal target area. In the current study, both transgenic and wild-type controls were evaluated in unconditioned (rotorod and locomotor activity) and conditioned (water maze and active avoidance) behavior tasks which tested hippocampal and striatal functioning. Compared to controls, the transgenic strain did not show differences in rotorod motor activity but did show a transient deficit in spatial navigation ability and a consistent impairment in active avoidance. The dominant-negative mutant receptor also resulted in a decrease in striatal dopamine and serotonin concentrations with no change in hippocampal monoamines. Collectively, these data suggest that animals expressing a truncated EphA5 receptor show deficits related to striatal functioning.

Regulation of EphB1 expression by dopamine signaling.

The Eph family tyrosine kinase receptors and their ligands have been implicated in axon guidance and neuronal migration during development of the nervous system. In the current study, we aim to characterize the nature of changes in EphB1 receptor expression following increases or decreases in dopamine activity. Neonatal mice (P3) were injected with 6-hydroxydopamine and allowed 13 days to recover. These animals show a profound depletion of dopamine in all areas assayed, with a corresponding dose-dependent decrease in EphB1 expression. Day 3 pups were also injected either chronically (P3-P16) or acutely (P3 only) with cocaine to determine how enhancing dopamine signaling would affect EphB1 signal density. It was found that both treatments significantly increased expression of EphB1 in the cortex, striatum and substantia nigra. Finally, animals were treated prenatally (E15-E17) with cocaine and sacrificed on P7. These animals also showed an increase in EphB1 signal density, but only in the dopaminergic terminal areas in the cortex and striatum. These studies indicate that dopamine activity regulates developmental expression of the tyrosine kinase receptor EphB1.

Altered sensitivity of CD81-deficient mice to neurobehavioral effects of cocaine.

CD81, also known as target of the antiproliferative antibody, is known to be expressed in astrocytes and involved in cell adhesion and, recently, we demonstrated its induction exclusively in the accumbens following cocaine. In the present study, the sensitivity of CD81-deficient mice to behavioral effects of cocaine was evaluated. It was found that CD81-deficient mice exhibited altered sensitivity to cocaine as assessed in the place preference conditioning paradigm and locomotor activity. This deficit in place preference conditioning was not accompanied by a deficit in acquisition or retention of water maze behavior. In addition, CD81 knockout mice exhibited higher levels of nucleus accumbens dopamine as compared to their controls. These observations are discussed in the context of the role of CD81 in cocaine-mediated behaviors.