Riding on the wind: volatile compounds dictate selection of grassland seedlings by snails.

Background and Aims: Seedling herbivory is an important selective filter in many plant communities. The removal of preferred food plants by both vertebrate and, more commonly, invertebrate herbivores can destroy entire seedling cohorts, and consequently dictate plant community assembly. Nevertheless, our understanding of how and why some seedlings are more prone to herbivore attack than their neighbours remains limited. For seedlings, where even minor tissue damage is fatal, avoiding contact with herbivores is probably advantageous and, on this basis, volatile organic compounds (VOCs) are strong candidates to fulfil a primary defensive role. Methods: We quantified seedling selection by snails (Cornu aspersum) for 14 common, European grassland species. Seedling acceptability was subsequently compared with species-specific expression of constitutive secondary defence metabolites (CSDMs), and VOCs to determine their relative influence on seedling selection. Results: We found no relationship between seedling acceptability and CSDMs, but seedling selection was strongly associated with VOC profiles. Monoterpenes (specifically ß-ocimene) were identified as likely attractants, while green leaf volatiles (GLVs) (3-hexen-1-ol acetate) were strongly associated with low seedling acceptability. Conclusions: By elucidating a relationship between VOCs and seedling acceptability, we contradict a long-held, but poorly tested, assumption that seedling selection by herbivores in (semi-)natural plant communities centres on CSDMs. Instead, our results corroborate recent work showing how GLVs, including 3-hexen-1-ol acetate, deter crop seedling selection by molluscs. Although our failure to establish any early-ontogenetic relationship between VOCs and CSDMs also suggests that the former do not 'advertise' possession of the latter, we nevertheless reveal the role that VOCs play in defending seedlings against herbivory before lethal damage occurs.

Tightening of the ATP-binding sites induces the opening of P2X receptor channels.

The opening of ligand-gated ion channels in response to agonist binding is a fundamental process in biology. In ATP-gated P2X receptors, little is known about the molecular events that couple ATP binding to channel opening. In this paper, we identify structural changes of the ATP site accompanying the P2X2 receptor activation by engineering extracellular zinc bridges at putative mobile regions as revealed by normal mode analysis. We provide evidence that tightening of the ATP sites shaped like open 'jaws' induces opening of the P2X ion channel. We show that ATP binding favours jaw tightening, whereas binding of a competitive antagonist prevents gating induced by this movement. Our data reveal the inherent dynamic of the binding jaw, and provide new structural insights into the mechanism of P2X receptor activation.

Optical control of an ion channel gate.

The powerful optogenetic pharmacology method allows the optical control of neuronal activity by photoswitchable ligands tethered to channels and receptors. However, this approach is technically demanding, as it requires the design of pharmacologically active ligands. The development of versatile technologies therefore represents a challenging issue. Here, we present optogating, a method in which the gating machinery of an ATP-activated P2X channel was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of ATP, thus revealing an agonist-independent, light-induced gating mechanism. We demonstrate photocontrol of neuronal activity by a light-gated, ATP-insensitive P2X receptor, providing an original tool devoid of endogenous sensitivity to delineate P2X signaling in normal and pathological states. These findings open new avenues to specifically activate other ion channels independently of their natural stimulus.

Ecological Engineering Approaches to Improve Hydraulic Properties of Infiltration Basins Designed for Groundwater Recharge.

Infiltration systems are increasingly used in urban areas for groundwater recharge. The reduction of sediment permeability by physical and/or biological processes is a major problem in management of infiltration systems often requiring expensive engineering operations for hydraulic performance maintenance. To reduce these costs and for the sake of sustainable development, we proposed to evaluate the ability of ecological engineering approaches to reduce the biological clogging of infiltration basins. A 36-day field-scale experiment using enclosures was performed to test the influences of abiotic (light reduction by shading) and biotic (introduction of the macrophyte Vallisneria spiralis (L.) or the gastropod Viviparus viviparus (Linnaeus, 1758)) treatments to limit benthic biofilm biomass and to maintain or even increase hydraulic performances. We coupled biological characterization of sediment (algal biomass, bacterial abundance, total organic carbon, total nitrogen, microbial enzymatic activity, photosynthetic activity, and photosystem II efficiency) with hydraulic conductivity measurements to assess the effects of treatments on sediment permeability. The grazer Viviparus viviparus significantly reduced benthic biofilm biomass and enhanced hydraulic conductivity. The other treatments did not produce significant changes in hydraulic conductivity although Vallisneria spiralis affected photosynthetic activity of biofilm. Finally, our results obtained with Viviparus viviparus are promising for the development of ecological engineering solutions to prevent biological fouling in infiltration systems.

Agonist trapped in ATP-binding sites of the P2X2 receptor.

ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent "tethering" reaction, covalent bonds between a synthesized ATP-derived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 Å in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATP-binding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.

Potential contribution of spinal interneurons to the etiopathogenesis of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) consists of a group of adult-onset fatal and incurable neurodegenerative disorders characterized by the progressive death of motor neurons (MNs) throughout the central nervous system (CNS). At first, ALS was considered to be an MN disease, caused by cell-autonomous mechanisms acting specifically in MNs. Accordingly, data from ALS patients and ALS animal models revealed alterations in excitability in multiple neuronal populations, including MNs, which were associated with a variety of cellular perturbations such as protein aggregation, ribonucleic acid (RNA) metabolism defects, calcium dyshomeostasis, modified electrophysiological properties, and autophagy malfunctions. However, experimental evidence rapidly demonstrated the involvement of other types of cells, including glial cells, in the etiopathogenesis of ALS through non-cell autonomous mechanisms. Surprisingly, the contribution of pre-motor interneurons (INs), which regulate MN activity and could therefore critically modulate their excitability at the onset or during the progression of the disease, has to date been severely underestimated. In this article, we review in detail how spinal pre-motor INs are affected in ALS and their possible involvement in the etiopathogenesis of the disease.

Chronic nicotine increases midbrain dopamine neuron activity and biases individual strategies towards reduced exploration in mice.

Long-term exposure to nicotine alters brain circuits and induces profound changes in decision-making strategies, affecting behaviors both related and unrelated to drug seeking and consumption. Using an intracranial self-stimulation reward-based foraging task, we investigated in mice the impact of chronic nicotine on midbrain dopamine neuron activity and its consequence on the trade-off between exploitation and exploration. Model-based and archetypal analysis revealed substantial inter-individual variability in decision-making strategies, with mice passively exposed to nicotine shifting toward a more exploitative profile compared to non-exposed animals. We then mimicked the effect of chronic nicotine on the tonic activity of dopamine neurons using optogenetics, and found that photo-stimulated mice adopted a behavioral phenotype similar to that of mice exposed to chronic nicotine. Our results reveal a key role of tonic midbrain dopamine in the exploration/exploitation trade-off and highlight a potential mechanism by which nicotine affects the exploration/exploitation balance and decision-making.

Probing the ionotropic activity of glutamate GluD2 receptor in HEK cells with genetically-engineered photopharmacology.

Glutamate delta (GluD) receptors belong to the ionotropic glutamate receptor family, yet they don't bind glutamate and are considered orphan. Progress in defining the ion channel function of GluDs in neurons has been hindered by a lack of pharmacological tools. Here, we used a chemo-genetic approach to engineer specific and photo-reversible pharmacology in GluD2 receptor. We incorporated a cysteine mutation in the cavity located above the putative ion channel pore, for site-specific conjugation with a photoswitchable pore blocker. In the constitutively open GluD2 Lurcher mutant, current could be rapidly and reversibly decreased with light. We then transposed the cysteine mutation to the native receptor, to demonstrate with high pharmacological specificity that metabotropic glutamate receptor signaling triggers opening of GluD2. Our results assess the functional relevance of GluD2 ion channel and introduce an optogenetic tool that will provide a novel and powerful means for probing GluD2 ionotropic contribution to neuronal physiology.

Neurotransmitters are chemicals released by the body that trigger activity in neurons. Receptors on the surface of neurons detect these neurotransmitters, providing a link between the inside and the outside of the cell. Glutamate is one of the major neurotransmitters and is involved in virtually all brain functions. Glutamate binds to two different types of receptors in neurons. Ionotropic receptors have pores known as ion channels, which open when glutamate binds. This is a fast-acting response that allows sodium ions to flow into the neuron, triggering an electrical signal. Metabotropic receptors, on the other hand, trigger a series of events inside the cell that lead to a response. Metabotropic receptors take more time than ionotropic receptors to elicit a response in the cell, but their effects last much longer. One type of receptor, known as the GluD family, is very similar to ionotropic glutamate receptors but does not directly respond to glutamate. Instead, the ion channel of GluD receptors opens after being activated by glutamate metabotropic receptors. GluD receptors are produced throughout the brain and play roles in synapse formation and activity, but the way they work remains unclear. An obstacle to understanding how GluD receptors work is the lack of molecules that can specifically block these receptors' ion channel activity. Lemoine et al. have developed a tool that enables control of the ion channel in GluD receptors using light. Human cells grown in the lab were genetically modified to produce a version of GluD2 (a member of the GluD family) with a light-sensitive molecule attached. In darkness or under green light, the light-sensitive molecule blocks the channel and prevents ions from passing through. Under violet light, the molecule twists, and ions can flow through the channel. With this control over the GluD2 ion channel activity, Lemoine et al. were able to validate previous research showing that the activation of metabotropic glutamate receptors can trigger GluD2 to open. The next step will be to test this approach in neurons. This will help researchers to understand what role GluD ion channels play in neuron to neuron communication.

Manipulating midbrain dopamine neurons and reward-related behaviors with light-controllable nicotinic acetylcholine receptors.

Dopamine (DA) neurons of the ventral tegmental area (VTA) integrate cholinergic inputs to regulate key functions such as motivation and goal-directed behaviors. Yet the temporal dynamic range and mechanism of action of acetylcholine (ACh) on the modulation of VTA circuits and reward-related behaviors are not known. Here, we used a chemical-genetic approach for rapid and precise optical manipulation of nicotinic neurotransmission in VTA neurons in living mice. We provide direct evidence that the ACh tone fine-tunes the firing properties of VTA DA neurons through ß2-containing (ß2*) nicotinic ACh receptors (nAChRs). Furthermore, locally photo-antagonizing these receptors in the VTA was sufficient to reversibly switch nicotine reinforcement on and off. By enabling control of nicotinic transmission in targeted brain circuits, this technology will help unravel the various physiological functions of nAChRs and may assist in the design of novel therapies relevant to neuropsychiatric disorders.

Field-based assessment of the mechanism of maize yield enhancement by Azospirillum lipoferum CRT1.

Plant Growth-Promoting Bacteria (PGPB) of the genus Azospirillum are known to enhance root growth and yield in many plant species including cereals. To probe the underlying mechanisms, correlations between modifications of yield and 6-leaf plantlet characteristics were estimated on maize in four fields with contrasting soil properties over two consecutive years using the commercial isolate A. lipoferum CRT1. In both years, plantlet metabolome, photosynthetic potential and organ morphology were found to display field- and inoculation-specific signatures. Metabolomic analyses revealed that A. lipoferum CRT1 mostly affected sugar metabolism with no suggested impact on N and P assimilation. Mineral nitrogen feeding increased yield but did not affect yield enhancement by the bacterial partner. However, greater improvements of leaf photosynthetic potential correlated with yield diminutions and larger plantlets in all of their proportions correlated with yield enhancements. Bacterial inoculation restored proper seed-to-adult plant ratio when it accidentally dropped below 80%. Only in these cases did it raise yield. All in all, securing mature plant density is hypothesized as being the primary driver of A. lipoferum CRT1-mediated yield enhancement in maize fields.

Optogenetic Control of Mammalian Ion Channels with Chemical Photoswitches.

In neurons, ligand-gated ion channels decode the chemical signal of neurotransmitters into an electric response, resulting in a transient excitation or inhibition. Neurotransmitters act on multiple receptor types and subtypes, with spatially and temporally precise patterns. Hence, understanding the neural function of a given receptor requires methods for its targeted, rapid activation/inactivation in defined brain regions. To address this, we have developed a versatile optochemical genetic strategy, which allows the reversible control of defined receptor subtypes in designated cell types, with millisecond and micrometer precision. In this chapter, we describe the engineering of light-activated and -inhibited neuronal nicotinic acetylcholine receptors, as well as their characterization and use in cultured cells.

Photo-switchable tweezers illuminate pore-opening motions of an ATP-gated P2X ion channel.

P2X receptors function by opening a transmembrane pore in response to extracellular ATP. Recent crystal structures solved in apo and ATP-bound states revealed molecular motions of the extracellular domain following agonist binding. However, the mechanism of pore opening still remains controversial. Here we use photo-switchable cross-linkers as 'molecular tweezers' to monitor a series of inter-residue distances in the transmembrane domain of the P2X2 receptor during activation. These experimentally based structural constraints combined with computational studies provide high-resolution models of the channel in the open and closed states. We show that the extent of the outer pore expansion is significantly reduced compared to the ATP-bound structure. Our data further reveal that the inner and outer ends of adjacent pore-lining helices come closer during opening, likely through a hinge-bending motion. These results provide new insight into the gating mechanism of P2X receptors and establish a versatile strategy applicable to other membrane proteins.

Exploring the ATP-binding site of P2X receptors.

P2X receptors are ATP-gated non-selective cation channels involved in many different physiological processes, such as synaptic transmission, inflammation, and neuropathic pain. They form homo- or heterotrimeric complexes and contain three ATP-binding sites in their extracellular domain. The recent determination of X-ray structures of a P2X receptor solved in two states, a resting closed state and an ATP-bound, open-channel state, has provided unprecedented information not only regarding the three-dimensional shape of the receptor, but also on putative conformational changes that couple ATP binding to channel opening. These data provide a structural template for interpreting the huge amount of functional, mutagenesis, and biochemical data collected during more than fifteen years. In particular, the interfacial location of the ATP binding site and ATP orientation have been successfully confirmed by these structural studies. It appears that ATP binds to inter-subunit cavities shaped like open jaws, whose tightening induces the opening of the ion channel. These structural data thus represent a firm basis for understanding the activation mechanism of P2X receptors.

Intermediate closed channel state(s) precede(s) activation in the ATP-gated P2X2 receptor.

The molecular mechanism underlying channel opening in response to agonist binding remains a challenging issue in neuroscience. In this regard, many efforts have been recently undertaken in ATP-gated P2X receptors. Among those efforts, we have provided evidence in the P2X2 receptor that tightening of ATP sites upon agonist binding induces opening of the ion channel. Here we extend our analysis to show that the sulfhydryl-reactive ATP analog 8-thiocyano-ATP (NCS-ATP), a potent P2X2 agonist, when covalently labeled in the ATP-binding site at position Leu186 likely favors the tightening mechanism, but not the channel opening mechanism. Our data predict the existence of intermediate or preactivation state(s) trapped by NCS-ATP, in which tightening of the binding site is favored while the channel is still closed. We propose that this (these) intermediate ATP-bound state(s) prime(s) channel gating in the P2X2 receptor.