Mood-related behavioral and neurochemical alterations in mice exposed to low chlorpyrifos levels during the brain growth spurt.

Organophosphates are among the most used pesticides. Particularly, chlorpyrifos (CPF) is responsible for a number of deleterious effects on brain development, which may program behavioral changes later in life. Here, we investigated whether a regimen of early low level CPF exposure that did not result in a significant inhibition of acetylcholinesterase (AChE) had deleterious effects on mood-related behaviors, as well as on cholinergic and serotonergic biomarkers in the mice brain. From the 3rd to 9th postnatal day (PN), male and female Swiss mice were subcutaneously injected with CPF. Mice were submitted to a battery of behavioral tests from PN60 to PN63: open field, elevated plus maze and forced swimming tests. The cholinergic and serotonergic biomarkers were assessed at PN10 and PN63. Our data indicated that early CPF exposure increased anxiety-like behavior in females and altered decision-making behavior in both sexes. Most biochemical alterations were sex-dependent and restricted to females. At PN10, CPF female mice showed increased serotonin and choline transporter binding in cerebral cortex. Distinctively, in adult females, the effects indicated a hypoactive state: CPF exposure reduced 5-HT1a receptor binding in cerebral cortex, as well as serotonin transporter binding and choline acetyltransferase activity in brainstem. Our results indicate that CPF exposure during the brain growth spurt deregulates serotonergic and cholinergic biomarkers. The effects are consistent with impaired synaptic function, may be related to long-term mood disorders and point out to higher female susceptibility.

Methamidophos exposure during the early postnatal period of mice: immediate and late-emergent effects on the cholinergic and serotonergic systems and behavior.

Organophosphates (OPs) are among the most used pesticides. Although some OPs have had their use progressively more restricted, other OPs are being used without sufficient investigation of their effects. Here, we investigated the immediate neurochemical and delayed neurochemical and behavioral actions of the OP methamidophos to verify whether there are concerns regarding exposure during early postnatal development. From the third to the nineth postnatal day (PN), Swiss mice were sc injected with methamidophos (1mg/kg). At PN10, we assessed cholinergic and serotonergic biomarkers in the cerebral cortex and brainstem. From PN60 to PN63, mice were submitted to a battery of behavioral tests and subsequently to biochemical analyses. At PN10, the effects were restricted to females and to the cholinergic system: Methamidophos promoted increased choline transporter binding in the brainstem. At PN63, in the brainstem, there was a decrease in choline transporter, a female-only decrease in 5HT1A and a male-only increase in 5HT2 receptor binding. In the cortex, choline acetyltransferase activity was decreased and 5HT2 receptor binding was increased both in males and females. Methamidophos elicited behavioral alterations, suggestive of increased depressive-like behavior and impaired decision making. There were no significant alterations on anxiety-related measures and on memory/learning. Methamidophos elicited cholinergic and serotonergic alterations that depended on brain region, sex, and age of the animals. These outcomes, together with the behavioral effects, indicate that this OP is deleterious to the developing brain and that alterations are indeed identified long after the end of exposure.

Exposure to methamidophos at adulthood adversely affects serotonergic biomarkers in the mouse brain.

Epidemiologic studies describe a potential risk of depression and suicide in farm workers exposed to organophosphates (OPs). In a previous study we observed an increase in depressive-like behavior in adult mice exposed to the OP pesticide methamidophos. Considering the association between depression and the serotonergic (5HT) system, in the present study we investigated whether a subchronic exposure to methamidophos affects the serotonergic system of adult mice. From postnatal day 60 to 89 (PN60 to PN89), one of two concentrations of methamidophos (higher dose: 5.25 µg/ml; lower dose: 1.31 µg/ml) or vehicle was administered in the drinking water of male Swiss mice. We evaluated three serotonergic biomarkers during (PN89) and after (PN100) the exposure period: 5HT(1A) receptor binding with [(3)H]OH-DPAT, 5HT(2) receptor binding with [(3)H]ketanserin and 5HT transporter binding with [(3)H]paroxetine. Methamidophos elicited robust decreases in binding for all 5HT markers. These decreases were evident in brain regions containing 5HT cell bodies and dendritic arbors (midbrain, brainstem) as well as in the cerebral cortex, which contains 5HT projections. In the cerebral cortex, effects were identified in mice exposed to the higher dose of methamidophos while in the midbrain and brainstem, both doses elicited significant effects. Overall, effects were present both during and after exposure, even though there were some regional disparities regarding the persistence of effects. Our results indicate that exposure to methamidophos affects synaptic transmission promoting decreases of specific serotonergic biomarkers. These data suggest a mechanism of action of this pesticide that might explain the increased depressive-like behavior in adult mice.

Exposure to nicotine and ethanol in adolescent mice: effects on depressive-like behavior during exposure and withdrawal.

Depression and use of addictive substances are two of the most frequent public health problems of adolescents. However, little is known about the association between depression and drug use. Considering that ethanol and nicotine are the most widely used and abused drugs by adolescents, here, we evaluated the depressive-like behavior of C57BL/6 male and female mice exposed to nicotine (NIC) and/or ethanol (ETOH) from the 30th to the 45th (PN30-45) postnatal day. Four groups were analyzed: 1) concomitant NIC (50µg/ml in 2% saccharin to drink) and ETOH (25%, 2g/kg i.p. injected every other day) exposure; 2) NIC exposure; 3) ETOH exposure; 4) vehicle. Immobile behavior, an animal model of depressive behavior, was assessed in the forced swimming test (FST) while the anhedonic state was assessed in the sucrose preference test (SPT) by the end of exposure (PN45-47) as well as during short- (PN50-52) and long-term (PN75-77) withdrawal. In the FST, ETOH female mice showed a reduction in immobility time by the end of exposure while, during long-term withdrawal, immobility time was increased. Short-term withdrawal elicited an increase in immobility time only in female NIC mice. In the SPT, males from both NIC and NIC+ETOH groups showed increased sucrose consumption, suggesting a reward-craving effect during short-term withdrawal. During long-term withdrawal, NIC male mice showed an anhedonic effect. Adolescent nicotine, ethanol and nicotine+ethanol combined exposures during adolescence thus elicit gender-selective effects both during exposure and withdrawal that may contribute to the increased prevalence of depression among drug users.

Exposure to methamidophos at adulthood elicits depressive-like behavior in mice.

Epidemiologic studies have suggested that organophosphate exposure is associated with an increased risk of depression and suicide. Considering that the neurobiological basis of this association is not well understood, in the present study we evaluated the depressive-like behavior of Swiss mice subchronically exposed to the organophosphate methamidophos at adulthood. From postnatal days 60 to 89 (PN60-PN89), one of two concentrations of methamidophos [higher dose (HiD): 5.25 microg/ml; lower dose (LoD): 1.31 microg/ml] or vehicle was administered in the drinking water. Immobile behavior, an animal model of depressive behavior, was assessed in the forced swimming and tail suspension tests either during (PN88) or after (PN99) the exposure period. On the subsequent day (PN89 or PN100), the Rota-rod and endurance swimming tests were used to evaluate motor performance. Brain acetylcholinesterase activity was quantified. During exposure, the LoD group presented increased immobility in the tail suspension test when compared to controls. The HiD group presented increased immobility in the forced swimming test when compared to LoD and control groups, an effect that emerged after cessation of exposure. There were no motor performance alterations. During exposure, acetylcholinesterase activity was inhibited in the HiD (43%) and LoD (15%) groups. After exposure, enzyme activity was reduced (25%) only in the HiD group. There were no signs of systemic toxicity. There were no correlations between acetylcholinesterase activity and behavioral measures. Our results indicate that methamidophos at doses below the threshold for systemic toxicity induces depressive-like behavior in adult mice.

Nicotine and ethanol interact during adolescence: effects on the central cholinergic systems.

Co-occurrence of tobacco smoking and alcohol consumption during adolescence is frequent and well documented. However, little is known about the basic neurobiology of the combined exposure in the adolescent brain. Since nicotine is a cholinergic agonist and it has been shown that ethanol interferes with nicotinic acetylcholine receptors (nAChRs), the current work focused on cholinergic systems. From the 30th to the 45th postnatal day (PN), C57BL/6 male and female mice were exposed to nicotine free base (NIC) and/or ethanol (ETOH). Four groups were analyzed: 1) concomitant NIC (50 microg/ml in 2% saccharin to drink) and ETOH (25%, 2 g/kg i.p. injected every other day) exposure; 2) NIC exposure; 3) ETOH exposure; 4) vehicle. We assessed nAChR binding, choline acetyltransferase (ChAT) activity and [3H]hemicholinium-3 (HC-3) binding in the cerebral cortex and midbrain of mice on PN45. In the cortex, ETOH had no effect on nAChRs. In contrast, NIC produced nAChR upregulation while NIC+ETOH elicited a more pronounced effect. In the midbrain, neither ETOH nor NIC had effects on nAChRs. NIC+ETOH, however, elicited a robust nAChR upregulation. Regarding ChAT activity, treatment effects differed between males and females in the cortex. Male NIC mice presented an increase in ChAT. However, ETOH reversed this effect. In contrast, female NIC mice presented decreased ChAT activity. In the midbrain, ETOH increased ChAT. HC-3 binding was not affected. These results indicate that the central cholinergic system is a site at which nicotine and ethanol interact. This interaction might underlie the association between tobacco and alcohol consumption during adolescence.

A minor beta-structured conformation is the active state of a fusion peptide of vesicular stomatitis virus glycoprotein.

Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in the VSV G protein, comprising the residues 145-164, directly involved in membrane interaction and fusion. In the present work we studied the interaction of pep[145-164] with membranes using NMR to solve the structure of the peptide in two membrane-mimetic systems: SDS micelles and liposomes composed of phosphatidylcholine and phosphatidylserine (PC:PS vesicles). The presence of medium-range NOEs showed that the peptide has a tendency to form N- and C-terminal helical segments in the presence of SDS micelles. Analysis of the chemical shift index indicated helix-coil equilibrium for the C-terminal helix under all conditions studied. At pH 7.0, the N-terminal helix also displayed a helix-coil equilibrium when pep[145-164] was free in solution or in the presence of PC:PS. Remarkably, at the fusogenic pH, the region of the N-terminal helix in the presence of SDS or PC:PS presented a third conformational species that was in equilibrium with the helix and random coil. The N-terminal helix content decreases pH and the minor beta-structured conformation becomes more prevalent at the fusogenic pH. These data point to a beta-conformation as the fusogenic active structure-which is in agreement with the X-ray structure, which shows a beta-hairpin for the region corresponding to pep[145-164].

Combined exposure to nicotine and ethanol in adolescent mice differentially affects memory and learning during exposure and withdrawal.

Human adolescents often associate tobacco smoking and consumption of alcoholic beverages. In spite of this frequent association, little is known about the basic neurobiology of the dual exposure in the adolescent brain. In the present work, we assessed, through the use of the step-through passive avoidance box (2mA, 2s; test-retest interval of 24h), short- and long-term memory/learning effects of nicotine (NIC) and/or ethanol (ETOH) exposure during adolescence (postnatal days 30-45: PN30-45) in four groups of male and female C57BL/6 mice: (1) concomitant NIC [nicotine free base solution (50microg/ml) in 2% saccharin to drink] and ETOH [ethanol solution (25%, 2g/kg) i.p. injected every other day] exposure; (2) NIC exposure; (3) ETOH exposure; (4) vehicle. During exposure (PN44-45), deficits in memory/learning due to concomitant NIC+ETOH exposure reflected the summation of the two individual sets of effects. During a short-term drug withdrawal (PN49-50), nicotine improved memory/learning, however, ethanol blocked nicotine-induced improvements. One month post-exposure (PN74-75), a significant female-only improvement in memory/learning was observed as a result of co-administration. In conclusion, our results suggest that detrimental effects of nicotine and ethanol on memory/learning during adolescent combined exposure represent a worsened outcome from the dual exposure. However, negative effects of the combined exposure fail to persist during withdrawal. In fact, the combined exposure elicits a sex-dependent late onset beneficial effect on memory/learning during withdrawal.

Membrane fusion induced by vesicular stomatitis virus depends on histidine protonation.

Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. VSV-induced membrane fusion occurs at a very narrow pH range, between 6.2 and 5.8, suggesting that His protonation is required for this process. To investigate the role of His in VSV fusion, we chemically modified these residues using diethylpyrocarbonate (DEPC). We found that DEPC treatment inhibited membrane fusion mediated by VSV in a concentration-dependent manner and that the complete inhibition of fusion was fully reversed by incubation of modified virus with hydroxylamine. Fluorescence measurements showed that VSV modification with DEPC abolished pH-induced conformational changes in G protein, suggesting that His protonation drives G protein interaction with the target membrane at acidic pH. Mass spectrometry analysis of tryptic fragments of modified G protein allowed the identification of the putative active His residues. Using synthetic peptides, we showed that the modification of His-148 and His-149 by DEPC, as well as the substitution of these residues by Ala, completely inhibited peptide-induced fusion, suggesting the direct participation of these His in VSV fusion.

Mayaro virus infection cycle relies on casein kinase 2 activity.

Replication of Mayaro virus in Vero cells induces dramatic cytopathic effects and cell death. In this study, we have evaluated the role of casein kinase 2 (CK2) during Mayaro virus infection cycle. We found that CK2 was activated during the initial stages of infection ( approximately 36% after 4h). This activation was further confirmed when the enzyme was partially purified from the cellular lysate either by Mono Q 5/5Hr column or heparin-agarose column. Using this later column, we found that the elution profile of CK2 activity from infected cells was different from that obtained for control cell enzyme, suggesting a structural modification of CK2 after infection. Treatment of infected cells with a cell-permeable inhibitor of CK2, dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB), abolished the cytopathic effect in a dose-dependent manner. Together this set of data demonstrates for the first time that CK2 activity in host cells is required in Mayaro virus infection cycle.