Can a mixture of agrochemicals (glyphosate, chlorpyrifos and chlorothalonil) mask the perception of an individual chemical? A hidden trap underlying ecological risk.

As aquatic environments associated with conventional agriculture are exposed to various pesticides, it is important to identify any possible interactions that modify their effects when in a mixture. We applied avoidance tests with Danio rerio, exposing juveniles to three relevant current use pesticides: chlorpyrifos (CPF), chlorothalonil (CTL) and glyphosate (Gly), individually and in binary mixtures (CPF-Gly and CTL-Gly). Our goal was to identify the potential of contaminants to trigger the avoidance response in fish and detect any changes to that response resulting from binary mixtures. Avoidance was assessed for three hours using an open gradient system with six levels of increasing concentrations. Fish avoided environmentally relevant concentrations of the three compounds. The avoidance of CPF [AC50 = 7.95 (3.3-36.3) µg/L] and CTL [AC50 = 3.41 (1.2-41.6) µg/L] was evident during the entire period of observation. In the case of Gly, the response changed throughout the experiment: initially (until 100 min) the fish tolerated higher concentrations of the herbicide [AC50 = 52.2 (12.1-2700) µg/L] while during the later period (after 100 min) a clearer avoidance [1.5 (0.8-4.2) µg/L] was observed. The avoidance recorded using CPF and CTL alone was attenuated by the presence of Gly. Applying an additive concentration model, Gly initially acted synergistically with the other two compounds, although this interaction was not observed during the later period. Avoidance gives us an idea of how the distribution of populations may be altered by contamination, our results suggest that in some mixtures this response may be inhibited, at least temporarily, thus masking the ecological risk of the exposure.

Functional and Structural Characterization of ClC-1 and Nav1.4 Channels Resulting from CLCN1 and SCN4A Mutations Identified Alone and Coexisting in Myotonic Patients.

Non-dystrophic myotonias have been linked to loss-of-function mutations in the ClC-1 chloride channel or gain-of-function mutations in the Nav1.4 sodium channel. Here, we describe a family with members diagnosed with Thomsen's disease. One novel mutation (p.W322*) in CLCN1 and one undescribed mutation (p.R1463H) in SCN4A are segregating in this family. The CLCN1-p.W322* was also found in an unrelated family, in compound heterozygosity with the known CLCN1-p.G355R mutation. One reported mutation, SCN4A-p.T1313M, was found in a third family. Both CLCN1 mutations exhibited loss-of-function: CLCN1-p.W322* probably leads to a non-viable truncated protein; for CLCN1-p.G355R, we predict structural damage, triggering important steric clashes. The SCN4A-p.R1463H produced a positive shift in the steady-state inactivation increasing window currents and a faster recovery from inactivation. These gain-of-function effects are probably due to a disruption of interaction R1463-D1356, which destabilizes the voltage sensor domain (VSD) IV and increases the flexibility of the S4-S5 linker. Finally, modelling suggested that the p.T1313M induces a strong decrease in protein flexibility on the III-IV linker. This study demonstrates that CLCN1-p.W322* and SCN4A-p.R1463H mutations can act alone or in combination as inducers of myotonia. Their co-segregation highlights the necessity for carrying out deep genetic analysis to provide accurate genetic counseling and management of patients.

Neurotoxicity of organophosphate pesticides could reduce the ability of fish to escape predation under low doses of exposure.

Biomarkers are frequently used in ecotoxicology as they allow to study toxicant effects happening at low concentrations of exposure. However, most sublethal studies only evaluate cellular biomarkers which lack evident ecological relevance. We used a multibiomarker approach to estimate the toxic effects of ethoprophos, an organophosphate insecticide commonly used in banana plantations, on the tropical fish Astyanax aeneus (Characidae). We measured biomarkers at sub-individual (cellular) and individual (metabolism, behavior) levels and examined relationships among these responses. A sublethal exposure to ethoprophos caused a significant (54%) reduction of brain Cholinesterase (ChE) activity, reflecting the pesticide's high neurotoxicity. However, other biomarkers like oxidative stress, biotransformation reactions, and resting metabolic rate were not affected. Exposure to ethoprophos modified antipredator behaviors such as escape response and detection avoidance (light/dark preference): exposed fish escaped slower from a simulated attack and preferred brighter areas in a novel tank. The relationship between ChE activity and reaction time suggests that pesticide-induced ChE inhibition reduces escape ability in fish. Our results provide evidence that impacts of organophosphate pesticides on fish ecological fitness can occur even with short exposures at very low concentrations.

Subconvulsant doses of pentylenetetrazol uncover the epileptic phenotype of cultured synapsin-deficient Helix serotonergic neurons in the absence of excitatory and inhibitory inputs.

Synapsins are a family of presynaptic proteins related to several processes of synaptic functioning. A variety of reports have linked mutations in synapsin genes with the development of epilepsy. Among the proposed mechanisms, a main one is based on the synapsin-mediated imbalance towards network hyperexcitability due to differential effects on neurotransmitter release in GABAergic and glutamatergic synapses. Along this line, a non-synaptic effect of synapsin depletion increasing neuronal excitability has recently been described in Helix neurons. To further investigate this issue, we examined the effect of synapsin knock-down on the development of pentylenetetrazol (PTZ)-induced epileptic-like activity using single neurons or isolated monosynaptic circuits reconstructed on microelectrode arrays (MEAs). Compared to control neurons, synapsin-silenced neurons showed a lower threshold for the development of epileptic-like activity and prolonged periods of activity, together with the occurrence of spontaneous firing after recurrent PTZ-induced epileptic-like activity. These findings highlight the crucial role of synapsin on neuronal excitability regulation in the absence of inhibitory or excitatory inputs.

Inhibition of afferent transmission in the feeding circuitry of aplysia: persistence can be as important as size.

We are studying afferent transmission from a mechanoafferent, B21, to a follower, B8. During motor programs, afferent transmission is regulated so that it does not always occur. Afferent transmission is eliminated when spike propagation in B21 fails, i.e., when spike initiation is inhibited in one output region-B21's lateral process. Spike initiation in the lateral process is inhibited by the B52 and B4/5 cells. Individual B52 and B4/5-induced inhibitory postsynaptic potentials (IPSPs) in B21 differ. For example, the peak amplitude of a B4/5-induced IPSP is four times the amplitude of a B52 IPSP. Nevertheless, when interneurons fire in bursts at physiological (i.e., low) frequencies, afferent transmission is most effectively reduced by B52. Although individual B52-induced IPSPs are small, they have a long time constant and summate at low firing frequencies. Once IPSPs summate, they effectively block afferent transmission. In contrast, individual B4/5-induced IPSPs have a relatively short time constant and do not summate at low frequencies. B52 and B4/5 therefore differ in that once synaptic input from B52 becomes effective, afferent transmission is continuously inhibited. In contrast, periods of B4/5-induced inhibition are interspersed with relatively long intervals in which inhibition does not occur. Consequently, the probability that afferent transmission will be inhibited is low. In conclusion, it is widely recognized that afferent transmission can be regulated by synaptic input. Our experiments are, however, unusual in that they relate specific characteristics of postsynaptic potentials to functional inhibition. In particular we demonstrate the potential importance of the IPSP time constant.

Feeding neural networks in the mollusc Aplysia.

Aplysia feeding is striking in that it is executed with a great deal of plasticity. At least in part, this flexibility is a result of the organization of the feeding neural network. To illustrate this, we primarily discuss motor programs triggered via stimulation of the command-like cerebral-buccal interneuron 2 (CBI-2). CBI-2 is interesting in that it can generate motor programs that serve opposing functions, i.e., programs can be ingestive or egestive. When programs are egestive, radula-closing motor neurons are activated during the protraction phase of the motor program. When programs are ingestive, radula-closing motor neurons are activated during retraction. When motor programs change in nature, activity in the radula-closing circuitry is altered. Thus, CBI-2 stimulation stereotypically activates the protraction and retraction circuitry, with protraction being generated first, and retraction immediately thereafter. In contrast, radula-closing motor neurons can be activated during either protraction or retraction. Which will occur is determined by whether other cerebral and buccal neurons are recruited, e.g. radula-closing motor neurons tend to be activated during retraction if a second CBI, CBI-3, is recruited. Fundamentally different motor programs are, therefore, generated because CBI-2 activates some interneurons in a stereotypic manner and other interneurons in a variable manner.