No abstract

The impact of sub-lethal doses of herbicides on human health and the environment is a matter of controversy. Due to the fact that evidence particularly of the effects of glyphosate on the central nervous system of rat offspring by in utero exposure is scarce, the purpose of the present study was to assess the neurobehavioral effects of chronic exposure to a glyphosate-containing herbicide during pregnancy and lactation. To this end, pregnant Wistar rats were exposed through drinking water to 0.2% or 0.4% of a commercial formulation of glyphosate (corresponding to a concentration of 0.65 or 1.30g/L of glyphosate, respectively) during pregnancy and lactation and neurobehavioral alterations in offspring were analyzed. The postnatal day on which each pup acquired neonatal reflexes (righting, cliff aversion and negative geotaxis) and that on which eyes and auditory canals were fully opened were recorded for the assessment of sensorimotor development. Locomotor activity and anxiety levels were monitored via open field test and plus maze test, respectively, in 45- and 90-day-old offspring. Pups exposed to a glyphosate-based herbicide showed early onset of cliff aversion reflex and early auditory canal opening. A decrease in locomotor activity and in anxiety levels was also observed in the groups exposed to a glyphosate-containing herbicide. Findings from the present study reveal that early exposure to a glyphosate-based herbicide affects the central nervous system in rat offspring probably by altering mechanisms or neurotransmitter systems that regulate locomotor activity and anxiety.

Cys-loop receptors mediate rapid transmission throughout the nervous system by converting a chemical signal into an electric one. They are pentameric proteins with an extracellular domain that carries the transmitter binding sites and a transmembrane region that forms the ion pore. Their essential function is to couple the binding of the agonist at the extracellular domain to the opening of the ion pore. How the structural changes elicited by agonist binding are propagated through a distance of 50 A to the gate is therefore central for the understanding of the receptor function. A step forward toward the identification of the structures involved in gating has been given by the recently elucidated high-resolution structures of Cys-loop receptors and related proteins. The extracellular-transmembrane interface has attracted attention because it is a structural transition zone where beta-sheets from the extracellular domain merge with alpha-helices from the transmembrane domain. Within this zone, several regions form a network that relays structural changes from the binding site toward the pore, and therefore, this interface controls the beginning and duration of a synaptic response. In this review, the most recent findings on residues and pairwise interactions underlying channel gating are discussed, the main focus being on the extracellular-transmembrane interface.

Each subunit in a homo-pentameric Cys-loop receptor contains a specialized coupling region positioned between the agonist binding domain and the ion conductive channel. To determine the contribution of each coupling region to the stability of the open channel, we constructed a receptor subunit (α7-5HT3A) with both a disabled coupling region and a reporter mutation that alters unitary conductance, and co-expressed normal and mutant subunits. The resulting receptors show single channel current amplitudes that are quantized according to the number of reporter mutations per receptor, allowing correlation of the number of intact coupling regions with mean open time. We find that each coupling region contributes an equal increment to the stability of the open channel. However by altering the numbers and locations of active coupling regions and binding sites, we find that a coupling region in a subunit flanked by inactive binding sites can still stabilize the open channel. We also determine minimal requirements for channel opening regardless of stability, and find that channel opening can occur in a receptor with one active coupling region flanked by functional binding sites, or with one active binding site flanked by functional coupling regions. The overall findings show that whereas the agonist binding sites contribute inter-dependently and asymmetrically to open channel stability, the coupling regions contribute independently and symmetrically.

Glyphosate-based herbicides (Gly-BHs) lead the world pesticide market. Although are frequently promoted as safe and of low toxicity, several investigations question its innocuousness. Previously, we described that oral exposure of rats to a Gly-BH during pregnancy and lactation decreased locomotor activity and anxiety in the offspring. The aim of the present study was to evaluate the mechanisms of neurotoxicity of this herbicide. Pregnant Wistar rats were supplied orally with 0.2 and 0.4% of Gly-BH (corresponding to 0.65 and 1.30 g/l of pure Gly, respectively) from gestational day (GD) 0, until weaning (postnatal day, PND, 21). Oxidative stress markers were determined in whole brain homogenates of PND90 offspring. The activity of acetylcholinesterase (AChE), transaminases, and alkaline phosphatase (AP) were assessed in prefrontal cortex (PFC), striatum, and hippocampus. Recognition memory was evaluated by the novel object recognition test. Brain antioxidant status was altered in Gly-BH-exposed rats. Moreover, AChE and transaminases activities were decreased and AP activity was increased in PFC, striatum and hippocampus by Gly-BH treatment. In addition, the recognition memory after 24 h was impaired in adult offspring perinatally exposed to Gly-BH. The present study reveals that exposure to a Gly-BH during early stages of rat development affects brain oxidative stress markers as well as the activity of enzymes involved in the glutamatergic and cholinergic systems. These alterations could contribute to the neurobehavioral variations reported previously by us, and to the impairment in recognition memory described in the present work.

Levamisole is an anthelmintic agent that exerts its therapeutic effect by acting as a full agonist of the nicotinic receptor (AChR) of nematode muscle. Its action at the mammalian muscle AChR has not been elucidated to date despite its wide use as an anthelmintic in humans and cattle. By single channel and macroscopic current recordings, we investigated the interaction of levamisole with the mammalian muscle AChR. Levamisole activates mammalian AChRs. However, single channel openings are briefer than those activated by acetylcholine (ACh) and do not appear in clusters at high concentrations. The peak current induced by levamisole is about 3% that activated by ACh. Thus, the anthelmintic acts as a weak agonist of the mammalian AChR. Levamisole also produces open channel blockade of the AChR. The apparent affinity for block (190 M at ؊70 mV) is similar to that of the nematode AChR, suggesting that differences in channel activation kinetics govern the different sensitivity of nematode and mammalian muscle to anthelmintics. To identify the structural basis of this different sensitivity, we performed mutagenesis targeting residues in the ␣ subunit that differ between vertebrates and nematodes. The replacement of the conserved ␣Gly-153 with the homologous glutamic acid of nematode AChR significantly increases the efficacy of levamisole to activate channels. Channel activity takes place in clusters having two different kinetic modes. The kinetics of the high open probability mode are almost identical when the agonist is ACh or levamisole. It is concluded that ␣Gly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs.At the neuromuscular junction, acetylcholine (ACh) 1 mediates fast neurotransmission by activating nicotinic receptors (AChRs). AChRs in nematode muscle are targets for anthelmintic chemotherapy. Levamisole and pyrantel are two widely used anthelmintic drugs. By binding to the AChR they lead to a depolarization of the somatic muscle of nematodes. The efficacy of these drugs is based on their ability to act as full agonists of AChRs in nematodes (1). Contractility and membrane potential measurements have shown that the nematode axial muscle is 10 -100 times more sensitive to the acute action of pyrantel and levamisole than the rat muscle (2). The molecular bases of this selectivity have not been yet elucidated. The kinetics of activation of nematode AChRs by levamisole has been studied in several preparations from parasite muscle (1, 3), but its action on mammalian muscle AChRs has not been described to date. The effects of levamisole on human neuronal ␣ 3 ␤ 2 and ␣ 3 ␤ 4 AChRs have been studied recently (4) with the voltage clamp method. It was shown that levamisole behaves as a weak partial agonist, an allosteric modulator, and an open channel blocker of neuronal AChRs (4).ACh is responsible for neuromuscular transmission in nematodes (1). In Caenorhabditis elegans muscle, levamisoleactivated AChRs are composed of the unc-38 subunit, which encodes an ␣ subunit, and lev-1 and...

No abstract

Nicotinic receptors (acetylcholine receptors, AChRs) play key roles in synaptic transmission throughout the nervous system. AChRs mediate neuromuscular transmission in nematodes, and they are targets for antiparasitic drugs. The anthelmintic agents levamisole and pyrantel, which are potent agonists of nematode muscle AChRs, are partial agonists of mammalian muscle AChRs. To further explore the structural basis of the differential activation of AChR subtypes by anthelmintics, we studied the activation of ␣7 AChRs using the high-conductance form of the ␣7-5-hydroxytryptamine-3A receptor, which is a good model for pharmacological studies involving the extracellular region of ␣7. Macroscopic and single-channel current recordings show that levamisole is a weak agonist of ␣7. It is interesting that pyrantel is a more potent agonist of ␣7 than acetylcholine (ACh). To identify determinants of this differential activation, we replaced residues of the complementary face of the binding site by the homologous residues in the muscle subunit and evaluated changes in activation. The mutation Q57G does not affect the activation by either ACh or levamisole. However, it increases EC 50 values and decreases the maximal response to pyrantel. Kinetic analysis shows that gating of the mutant channel activated by pyrantel is profoundly impaired. The decreased sensitivity of ␣7-Q57G to pyrantel agrees with its weak action at muscle AChRs, indicating that when glycine occupies position 57, as in the mammalian muscle AChR, pyrantel behaves as a partial agonist. Thus, position 57 located at the complementary face of the binding site plays a key role in the selective activation of AChRs by pyrantel.Cysteine-loop receptors are pentameric ligand-gated ion channels that play key roles in chemical synapses throughout the nervous system. In vertebrates, the cysteine-loop receptor superfamily consists of two families of cation-selective channels: the nicotinic acetylcholine (AChR) and the 5-hydroxytryptamine type 3 receptors (5HT 3 Rs); and two families of anionic channels: the ␥-aminobutyric acid and the glycine receptors (Le Novére and Changeux, 2001;Lester et al., 2004). In invertebrates, this superfamily also includes ␥-aminobutyric acid-gated cationic channels and ACh-, serotonin-, glutamate-, and histamine-gated chloride channels (Jones and Sattelle, 2003;Putrenko et al., 2005).AChR subunits are classified as ␣, which contain a disulfide bridge involved in the recognition and binding of agonists, and non-␣ subunits, which lack this motif. To date, several ␣ and non-␣ subunits have been identified. Receptors can be either heteromeric, composed of ␣ and non-␣ subunits, or homomeric, composed of five identical ␣ subunits. Each subunit is divided into an N-terminal or ligand-binding domain and a transmembrane region. The binding sites are located at the interface between two subunits (Brejc et al., 2001;Sine, 2002). Results from affinity labeling and sitedirected mutagenesis studies on AChR, which were later supported by the structural...