Single and Repeated Administration of Methylphenidate Modulates Synaptic Plasticity in Opposite Directions via Insertion of AMPA Receptors in Rat Hippocampal Neurons

Methylphenidate (MPH) is widely used in the treatment of Attention Deficit Hyperactivity Disorder. Several lines of evidence support that MPH can modulate learning and memory processes in different ways including improvement and impairment of test performances. A relevant factor in the efficacy of treatment is whether administration is performed once or several times. In this study we demonstrate opposite effects of MPH on performance of preadolescent rats in the Morris Water Maze test. Animals treated with a single dose (1 mg/kg) performed significantly better compared to controls, while in animals treated with repetitive administration at the same concentration performance was reduced. We found that hippocampal LTP in slices from rats treated with a single dose was increased, while LTP from rats treated with repetitive injections of MPH was lower than in controls. Using Western blot of CA1 areas from potentiated slices of rats treated with a single dose we found a significant increase of phosphorylation at Ser845 of GluA1 subunits, associated to an increased insertion of GluA1-containing AMPARs in the plasma membrane. These receptors were functional, because AMPA-dependent EPSCs recorded on CA1 were enhanced, associated to a significant increase in short-term plasticity. In contrast, CA1 samples from rats injected with MPH during six consecutive days, showed a significant decrease in the phosphorylation at Ser845 of GluA1 subunits associated to a lower insertion of GluA1-containing AMPARs. Accordingly, a reduction of the AMPA-mediated EPSCs and short-term plasticity was also observed. Taken together, our results demonstrate that single and repeated doses with MPH can induce opposite effects at behavioral, cellular, and molecular levels. The mechanisms demonstrated here in preadolescent rats are relevant to understand the effects of this psychostimulant in the treatment of ADHD

Intracellular pH regulation in rat round spermatids

Intracellular pH has been shown to be an important physiological parameter in cell cycle control and differentiation, aspects that are central to the spermatogenic process. However, the pH regulatory mechanisms in spermatogenic cells have not been systematically explored. In this work, measuring intracellular pH (pH(i)) with a fluorescent probe (BCECF), membrane potential with a fluorescent lipophilic anion (bisoxonol), and net movement of acid using a pH-stat system, we have found that rat round spermatids regulate pH(i) by means of a V-type H+-ATPase, a HCO3- entry pathway, a Na+/HCO3- dependent transport system, and a putative proton conductive pathway. Rat spermatids do not have functional base extruder transport systems. These pH regulatory characteristics seem specially designed to withstand acid challenges, and can generate sustained alkalinization upon acid exit stimulation

In Vivo Sub-chronic Treatment with Dichlorvos in Young Rats Promotes Synaptic Plasticity and Learning by a Mechanism that Involves Acylpeptide Hydrolase Instead of Acetylcholinesterase Inhibition. Correlation with Endogenous β-Amyloid Levels

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fphar.2017.00483/full#supplementary-materialAcylpeptide hydrolase (APEH) is a serine hydrolase that displays two catalytic activities, acting both as an exopeptidase toward short N-acylated peptides and as an endopeptidase toward oxidized peptides or proteins. It has been demonstrated that this enzyme can degrade monomers, dimers, and trimers of the Aβ1-40 peptide in the conditioned media of neuroblastoma cells. In a previous report, we showed that the specific inhibition of this enzyme by the organophosphate molecule dichlorvos (DDVP) triggers an enhancement of long-term potentiation in rat hippocampal slices. In this study, we demonstrate that the same effect can be accomplished in vivo by sub-chronic treatment of young rats with a low dose of DDVP (0.1 mg/kg). Besides exhibiting a significant enhancement of LTP, the treated animals also showed improvements in parameters of spatial learning and memory. Interestingly, higher doses of DDVP such as 2 mg/kg did not prove to be beneficial for synaptic plasticity or behavior. Due to the fact that at 2 mg/kg we observed inhibition of both APEH and acetylcholinesterase, we interpret that in order to achieve positive effects on the measured parameters only APEH inhibition should be obtained. The treatment with both DDVP doses produced an increase in the endogenous concentration of Aβ1-40, although this was statistically significant only at the dose of 0.1 mg/kg. We propose that APEH represents an interesting pharmacological target for cognitive enhancement, acting through the modulation of the endogenous concentration of Aβ1-40.CONICYT FONDECYT 1140856 (RS and FP), 11121645 (RS), 3140437 (FG

Protein kinase CK2 in postsynaptic densities: Phosphorylation of PSD-95/SAP90 and NMDA receptor regulation

Protein kinase CK2 (CK2) is highly expressed in rat forebrain where its function is not well understood. Subcellular distribution studies showed that the catalytic subunit of CK2 (CK2α) was enriched in postsynaptic densities (PSDs) by 68%. We studied the putative role of CK2 activity on N-methyl-d-aspartate receptor (NMDAR) function using isolated, patch-clamped PSDs in the presence of 2 mM extracellular Mg 2+. The usual activation by phosphorylation of the NMDARs in the presence of ATP was inhibited by the selective CK2 inhibitor 5,6-dichloro-1-β-ribofuranosyl benzimidazole (DRB). This inhibition was voltage-dependent, i.e., 100% at positive membrane potentials, while at negative potentials, inhibition was incomplete. Endogenous CK2 substrates were characterized by their ability to use GTP as a phosphoryl donor and susceptibility to inhibition by DRB. Immunoprecipitation assays and 2D gels indicated that PSD-95/SAP90, the NMDAR scaffolding protein, was a CK2 substrate, while the NR2A

In Vivo Sub-chronic Treatment with Dichlorvos in Young Rats Promotes Synaptic Plasticity and Learning by a Mechanism that Involves Acylpeptide Hydrolase Instead of Acetylcholinesterase Inhibition. Correlation with Endogenous β-Amyloid Levels

Acylpeptide hydrolase (APEH) is a serine hydrolase that displays two catalytic activities, acting both as an exopeptidase toward short N-acylated peptides and as an endopeptidase toward oxidized peptides or proteins. It has been demonstrated that this enzyme can degrade monomers, dimers, and trimers of the Aβ1-40 peptide in the conditioned media of neuroblastoma cells. In a previous report, we showed that the specific inhibition of this enzyme by the organophosphate molecule dichlorvos (DDVP) triggers an enhancement of long-term potentiation in rat hippocampal slices. In this study, we demonstrate that the same effect can be accomplished in vivo by sub-chronic treatment of young rats with a low dose of DDVP (0.1 mg/kg). Besides exhibiting a significant enhancement of LTP, the treated animals also showed improvements in parameters of spatial learning and memory. Interestingly, higher doses of DDVP such as 2 mg/kg did not prove to be beneficial for synaptic plasticity or behavior. Due to the fact that at 2 mg/kg we observed inhibition of both APEH and acetylcholinesterase, we interpret that in order to achieve positive effects on the measured parameters only APEH inhibition should be obtained. The treatment with both DDVP doses produced an increase in the endogenous concentration of Aβ1-40, although this was statistically significant only at the dose of 0.1 mg/kg. We propose that APEH represents an interesting pharmacological target for cognitive enhancement, acting through the modulation of the endogenous concentration of Aβ1-40

Differential Distribution and Activity Profile of Acylpeptide Hydrolase in the Rat Seminiferous Epithelium

Acylpeptide hydrolase (APEH) is a serine protease involved in amino acid recycling from acylated peptides (exopeptidase activity) and degradation of oxidized proteins (endoproteinase activity). This enzyme is inhibited by dichlorvos (DDVP), an organophosphate compound used as an insecticide. The role of APEH in spermatogenesis has not been established; therefore, the aim of this study was to characterize the distribution and activity profile of APEH during this process. For this purpose, cryosections of male reproductive tissues (testis and epididymis) and isolated cells (Sertoli cells, germ cells, and spermatozoa) were obtained from adult rats in order to analyze the intracellular localization of APEH by indirect immunofluorescence. In addition, the catalytic activity profiles of APEH in the different male reproductive tissues and isolated cells were quantified. Our results show that APEH is homogeneously distributed in Sertoli cells and early germ cells (spermatocytes and round spermatids), but this pattern changes during spermiogenesis. Specifically, in elongated spermatids and spermatozoa, APEH was localized in the acrosome and the principal piece. The exopeptidase activity was higher in the germ cell pool, compared to sperm and Sertoli cells, while the endoproteinase activity in epididymal homogenates was higher compared to testis homogenates at 24 h of incubation. In isolated cells, this activity was increased in Sertoli and germ cell pools, compared to spermatozoa. Taken together, these results indicate that APEH is differentially distributed in the testicular epithelium and undergoes re-localization during spermiogenesis. A possible role of APEH as a component of a protection system against oxidative stress and during sperm capacitation is discussed

Neurological and neuropsychological deterioration in artisanal gold miners from the town of Andacollo, Chile

It is widely known that human exposure to mercury vapor can cause neurological and neuropsychological deterioration. We have investigated if a population of Chilean artisanal gold-mining workers heavily exposed to elemental mercury (Hg0) display neurological and neuropsychological impairment. Male volunteers occupationally exposed to Hg0 ("gold miners", n = 35) were recruited and compared with a group of unexposed workers (n = 40). Blood specimens were obtained from both groups for total mercury quantification. Upon neurological examination, 71% of the "gold miners" group showed abnormalities expressed as frontal impairment, tremor, or simultaneously frontal impairment, parkinsonism, and pyramidal syndrome. In contrast, only 16% of the individuals in the control group displayed neurological abnormalities. The "gold miners" group also showed impairment of the neuropsychological performance and the distribution of abnormal scores for almost all the neuropsychological tests applied was significantly higher in this group compared to the control group. Educational level, alcohol intake, and smoking habits did not influence the outcome of the neurological or neuropsychological examinations. Finally, despite that the higher blood mercury concentrations were found in the “gold miners” group, they were not significantly different from the values obtained in the control group

Possible involvement of plasma antioxidant defences in training-associated decrease of platelet reponsiveness in humans

Artículo de publicación ISIHealth effects of pesticides are easily diagnosed when acute poisonings occurs, nevertheless, consequences from chronic exposure can only be observed when neuropsychiatric, neurodegenerative or oncologic pathologies appear. Therefore, early monitoring of this type of exposure is especially relevant to avoid the consequences of pathologies previously described; especially concerning workers exposed to pesticides on the job. For acute organophosphate pesticides (OPP) exposure, two biomarkers have been validated: plasma cholinesterase (ChE) and acetylcholinesterase (AChE) from erythrocytes. These enzymes become inhibited when people are exposed to high doses of organophosphate pesticides, along with clear signs and symptoms of acute poisoning; therefore, they do not serve to identify risk from chronic exposure. This study aims to assess a novel biomarker that could reflect neuropsychological deterioration associated with long-term exposure to organophosphate pesticides via the enzyme acylpeptide-hydrolase (ACPH), which has been recently identified as a direct target of action for some organophosphate compounds.The project Fondef D09I1057 was founded by FONDEF Fondo de Fomento al Desarrollo Científico y Tecnológico, CONICYT Chile (Fund for the promotion of scientific and technological development, National Commission for Scientific and Technological Research, Chile)