Bernardo Morales

BDNF regulates the maturation of inhibition and the critical period of plasticity in mouse visual cortex

Maturation of the visual cortex is influenced by visual experience during an early postnatal period. The factors that regulate such a critical period remain unclear. We examined the maturation and plasticity of the visual cortex in transgenic mice in which the postnatal rise of brain-derived neurotrophic factor (BDNF) was accelerated. In these mice, the maturation of GABAergic innervation and inhibition was accelerated. Furthermore, the age-dependent decline of cortical long-term potentiation induced by white matter stimulation, a form of synaptic plasticity sensitive to cortical inhibition, occurred earlier. Finally, transgenic mice showed a precocious development of visual acuity and an earlier termination of the critical period for ocular dominance plasticity. We propose that BDNF promotes the maturation of cortical inhibition during early postnatal life, thereby regulating the critical period for visual cortical plasticity.

Visual cortex is rescued from the effects of dark rearing by overexpression of BDNF

Visual deprivation such as dark rearing (DR) prolongs the critical period for ocular dominance plasticity and retards the maturation of γ-aminobutyric acid (GABA)ergic inhibition in visual cortex. The molecular signals that mediate the effects of DR on the development of visual cortex are not well defined. To test the role of brain-derived neurotrophic factor (BDNF), we examined the effects of DR in transgenic mice in which BDNF expression in visual cortex was uncoupled from visual experience and remained elevated during DR. In dark-reared transgenic mice, visual acuity, receptive field size of visual cortical neurons, critical period for ocular dominance plasticity, and intracortical inhibition were indistinguishable from those observed in light-reared mice. Therefore, BDNF overexpression is sufficient for the development of aspects of visual cortex in the absence of visual experience. These results suggest that reduced BDNF expression contributes to retarded maturation of GABAergic inhibition and delayed development of visual cortex during visual deprivation.

Zinc and Copper Modulate Differentially the P2X4 Receptor

Abstract: The rat ATP P2X4 receptor was expressed in Xenopus laevis oocytes to assess the effect of zinc and copper as possible regulators of purinergic mechanisms. ATP applied for 20 s evoked an inward cationic current with a median effective concentration (EC50) of 21.4 ± 2.8 μM and a Hill coefficient (nH) of 1.5 ± 0.1. Coapplication of ATP plus 10 μM zinc displaced leftward, in a parallel fashion, the ATP concentration‐response curve, reducing the EC50 to 8.4 ± 1.8 μM (p < 0.01) without altering the receptor nH. The zinc potentiation was fast in onset, easily reversible, and voltage‐independent and did not require metal preexposure. The zinc EC50 was 2‐5 μM, with a bell‐shaped curve. At concentrations of 100‐300 μM, zinc produced less potentiation, and at 1 mM, it inhibited 50% the ATP current. The effect of zinc was mimicked by cadmium. In contrast, copper inhibited the ATP‐evoked currents in a time‐ and concentration‐dependent fashion, reducing the maximal current (Imax) without altering the EC50. The copper‐induced inhibition was slow in onset, slowly reversible, and voltage‐independent. Whereas coapplication of 300 μM copper plus ATP reduced Imax to 36.2 ± 5%, the coapplication of, or 60‐s preexposure by, 10 μM copper reduced Imax to 79 ± 9.2% (p < 0.05) and 39.6 ± 8.7% (p < 0.01), respectively. The inhibition was noncompetitive in nature and mimicked by mercury. Cobalt, barium, and manganese did not modify significantly the ATP‐evoked current, demonstrating metal specificity. The simultaneous 1‐min preapplication of both metals revealed that the 10 μM zinc‐induced potentiation was obliterated by 10 μM copper, whereas 30 μM copper not only reduced the potentiation, but inhibited the ATP response. Following coapplication of both metals for 20 s with ATP, at least 100 μM copper was required to counteract the 10 μM zinc‐induced potentiation. The simultaneous preincubation with both metals provided evidence for a noncompetitive interaction. We hypothesize the existence of metal binding site(s), which are most likely localized in the extracellular domain of the P2X4 receptor structure. These sites are selective and accessible to extracellular metal applications and bind micromolar concentrations of metals. The present results are compatible with the working hypothesis that trace metals, such as copper and zinc, are physiological modulators of the P2X4 receptor. The modulation of brain purinergic transmission by physiologically and toxicologically relevant trace metal cations is highlighted.

Cross Talk among Calcium, Hydrogen Peroxide, and Nitric Oxide and Activation of Gene Expression Involving Calmodulins and Calcium-Dependent Protein Kinases in Ulva compressa Exposed to Copper Excess

To analyze the copper-induced cross talk among calcium, nitric oxide (NO), and hydrogen peroxide (H2O2) and the calcium-dependent activation of gene expression, the marine alga Ulva compressa was treated with the inhibitors of calcium channels, ned-19, ryanodine, and xestospongin C, of chloroplasts and mitochondrial electron transport chains, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and antimycin A, of pyruvate dehydrogenase, moniliformin, of calmodulins, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide, and of calcium-dependent protein kinases, staurosporine, as well as with the scavengers of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and of H2O2, ascorbate, and exposed to a sublethal concentration of copper (10 μm) for 24 h. The level of NO increased at 2 and 12 h. The first peak was inhibited by ned-19 and 3-(2,3-dichlorophenyl)-1,1-dimethylurea and the second peak by ned-19 and antimycin A, indicating that NO synthesis is dependent on calcium release and occurs in organelles. The level of H2O2 increased at 2, 3, and 12 h and was inhibited by ned-19, ryanodine, xestospongin C, and moniliformin, indicating that H2O2 accumulation is dependent on calcium release and Krebs cycle activity. In addition, pyruvate dehydrogenase, 2-oxoxglutarate dehydrogenase, and isocitrate dehydrogenase activities of the Krebs cycle increased at 2, 3, 12, and/or 14 h, and these increases were inhibited in vitro by EGTA, a calcium chelating agent. Calcium release at 2, 3, and 12 h was inhibited by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and ascorbate, indicating activation by NO and H2O2. In addition, the level of antioxidant protein gene transcripts decreased with N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide and staurosporine. Thus, there is a copper-induced cross talk among calcium, H2O2, and NO and a calcium-dependent activation of gene expression involving calmodulins and calcium-dependent protein kinases.

Absence of Long-Term Depression in the Visual Cortex of Glutamic Acid Decarboxylase-65 Knock-Out Mice

Long-term depression (LTD) is widely considered a mechanism for experience-induced synaptic weakening in the brain. Recent in vivo studies on glutamic acid decarboxylase [GAD 65 (−/−)] knock-out mice indicates that GABAergic synaptic inhibition is also required for the normal weakening of deprived inputs in the visual cortex. To better understand how GABAergic inhibition might control plasticity, we assessed the status of synaptic inhibition and LTD in visual cortical slices of GAD 65 knock-out mice. We found the following: (1) the efficacy of GABAergic synapses during repetitive activation is reduced in GAD 65 (−/−) mice; (2) the induction of LTD is impaired in the visual cortex of GAD 65 (−/−) mice; and (3) chronic, but not acute, treatment with the benzodiazepine agonist diazepam restores LTD in GAD 65 (−/−) mice. These results suggest that a certain inhibitory tone is required for the induction of LTD in visual cortex. We propose that the lack of visual cortical LTD in GAD 65 (−/−) may account for the lack of experience-dependent plasticity in these mice.

Co‐occurring increases of calcium and organellar reactive oxygen species determine differential activation of antioxidant and defense enzymes in Ulva compressa (Chlorophyta) exposed to copper excess

In order to analyse copper-induced calcium release and (reactive oxygen species) ROS accumulation and their role in antioxidant and defense enzymes activation, the marine alga Ulva compressa was exposed to 10 µM copper for 7 d. The level of calcium, extracellular hydrogen peroxide (eHP), intracellular hydrogen peroxide (iHP) and superoxide anions (SA) as well as the activities of ascorbate peroxidase (AP), glutathione reductase (GR), glutathione-S-transferase (GST), phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX) were determined. Calcium release showed a triphasic pattern with peaks at 2, 3 and 12 h. The second peak was coincident with increases in eHP and iHP and the third peak with the second increase of iHP. A delayed wave of SA occurred after day 3 and was not accompanied by calcium release. The accumulation of iHP and SA was mainly inhibited by organellar electron transport chains inhibitors (OETCI), whereas calcium release was inhibited by ryanodine. AP activation ceased almost completely after the use of OETCI. On the other hand, GR and GST activities were partially inhibited, whereas defense enzymes were not inhibited. In contrast, PAL and LOX were inhibited by ryanodine, whereas AP was not inhibited. Thus, copper stress induces calcium release and organellar ROS accumulation that determine the differential activation of antioxidant and defense enzymes.

Zinc enhances long‐term potentiation through P2X receptor modulation in the hippocampal CA1 region

Zn2+ is an essential ion that is stored in and co‐released from glutamatergic synapses and it modulates neurotransmitter receptors involved in long‐term potentiation (LTP). However, the mechanism(s) underlying Zn2+‐induced modulation of LTP remain(s) unclear. As the purinergic P2X receptors are relevant targets for Zn2+ action, we have studied their role in LTP modulation by Zn2+ in the CA1 region of rat hippocampal slices. Induction of LTP in the presence of Zn2+ revealed a biphasic effect – 5–50 μm enhanced LTP induction, whereas 100–300 μm Zn2+ inhibited LTP. The involvement of a purinergic mechanism is supported by the fact that application of the P2X receptor antagonists 2′,3′‐O‐(2,4,6‐trinitrophenyl) ATP (TNP‐ATP) and periodate‐oxidized ATP fully abolished the facilitatory effect of Zn2+. Notably, application of the P2X7 receptor‐specific antagonist Brilliant Blue G did not modify the Zn2+‐dependent facilitation of LTP. Exogenous ATP also produced a biphasic effect – 0.1–1 μm ATP facilitated LTP, whereas 5–10 μm inhibited LTP. The facilitatory effect of ATP was abolished by the application of TNP‐ATP and was modified in the presence of 5 μm Zn2+, suggesting that P2X receptors are involved in LTP induction and that Zn2+ leads to an increase in the affinity of P2X receptors for ATP. The latter confirms our previous results from heterologous expression systems. Collectively, our results indicate that Zn2+ at low concentrations enhances LTP by modulating P2X receptors. Although it is not yet clear which purinergic receptor subtype(s) is responsible for these effects on LTP, the data presented here suggest that P2X4 but not P2X7 is involved.

Noncholinesterase effects induced by organophosphate pesticides and their relationship to cognitive processes: implication for the action of acylpeptide hydrolase.

Organophosphate pesticides have been classically described as inhibitors of acetylcholinesterase (AChE) activity in insects and invertebrates. However, there is now more evidence supporting the hypothesis that these compounds also act through noncholinergic pathways, especially those related to cognitive processes. The enzyme acylpeptide hydrolase was identified as a new target for organophosphate pesticides. This enzyme is more sensitive than AChE to some organophosphates (OP), including dichlorvos, which is the parent compound for metrifonate, a therapeutic agent used in the treatment of cognitive impairment associated to Alzheimer´s disease. Therefore, there is some doubt as to whether the mechanism of action of this drug is mediated by a potentiation of cholinergic transmission. However, the direct action of acylpeptide hydrolase in cognitive processes and the physiological and molecular mechanisms underlying subacute exposure to OP have yet to be demonstrated. This review deals with evidence demonstrating the existence of mechanisms of actions of OP, which are independent of cholinergic pathway potentiation and which have an effect on cognitive processes. In addition, the possible participation of the enzyme acylpeptide hydrolase in these processes is also discussed. Finally, the possibility of using this enzyme activity as a new biomarker for exposure to OP is considered.

Platelet activating factor increases ciliary activity in the hamster oviduct through epithelial production of prostaglandin E2.

Abstract. We investigated the signal transduction mechanisms associated with an increase in ciliary beat frequency (CBF) produced by platelet activating factor (PAF) in oviductal ciliated cell cultures. In the range of concentrations similar to that produced by preimplantation embryos, PAF increased the CBF in a dose-dependent manner. The addition of PAF and prostaglandin E2 (PGE2) to the cultures produced a synergic increase of ciliary beating, suggesting that PAF and PGE2 signal transduction pathways may be associated. To demonstrate this hypothesis, cyclooxygenase-2 (COX-2) was selectively blocked by a specific inhibitor, NS-398, and the PAF-induced CBF increase was abolished. Moreover, a phospholipase A2 (PLA2) inhibitor, AACOCF3, blocks the PAF-induced CBF increase. PGE2 production by oviductal epithelial cells is stimulated by PAF, and WEB-2086, a PAF-receptor blocker, specifically blocks the PAF-induced PGE2 production. Using the fluorescent indicator fura-2, we measured the effect of PAF on intracellular Ca2+ concentration ([Ca2+]i) in individual ciliated cells. PAF induced a transient increase of [Ca2+]i that was blocked by WEB-2086 or by removal of extracellular Ca2+. We propose a mechanism for PAF-mediated signal transduction in the ciliated cells of the oviductal epithelium. Minimal doses of PAF trigger Ca2+ mobilization in tandem with increased PLA2 activity and a COX-2-mediated increase in PGE2. Local PGE2 production by the oviductal mucosa suggests the presence of an autocrine loop controlling ciliary activity.

MDMA ("ecstasy") impairs learning in the Morris Water Maze and reduces hippocampal LTP in young rats.

3,4-Methylenedioxymethamphetamine (MDMA), an important recreational psychostimulant drug, was examined for its ability to alter visuo-spatial learning and synaptic plasticity. Young rats received MDMA (0.2 and 2mg/kg s.c.) twice per day for 6 days while their visuo-spatial learning was tested using the Morris Water Maze. After this, animals were sacrificed and LTP induced in hippocampal slices. Visuo-spatial learning was impaired and LTP reduced, both dose-dependently, without changes in serotonin levels or paired-pulse facilitation. We conclude that low, nontoxic doses of MDMA, applied during several days, slow learning by impairing postsynaptic plasticity.