AutoEPG: Software for the Analysis of Electrical Activity in the Microcircuit Underpinning Feeding Behaviour of Caenorhabditis elegans

Background

The pharyngeal microcircuit of the nematode Caenorhabditis elegans serves as a model for analysing neural network activity and is amenable to electrophysiological recording techniques. One such technique is the electropharyngeogram (EPG) which has provided insight into the genetic basis of feeding behaviour, neurotransmission and muscle excitability. However, the detailed manual analysis of the digital recordings necessary to identify subtle differences in activity that reflect modulatory changes within the underlying network is time consuming and low throughput. To address this we have developed an automated system for the high-throughput and discrete analysis of EPG recordings (AutoEPG).

Methodology/Principal Findings

AutoEPG employs a tailor made signal processing algorithm that automatically detects different features of the EPG signal including those that report on the relaxation and contraction of the muscle and neuronal activity. Manual verification of the detection algorithm has demonstrated AutoEPG is capable of very high levels of accuracy. We have further validated the software by analysing existing mutant strains with known pharyngeal phenotypes detectable by the EPG. In doing so, we have more precisely defined an evolutionarily conserved role for the calcium-dependent potassium channel, SLO-1, in modulating the rhythmic activity of neural networks.

Conclusions/Significance

AutoEPG enables the consistent analysis of EPG recordings, significantly increases analysis throughput and allows the robust identification of subtle changes in the electrical activity of the pharyngeal nervous system. It is anticipated that AutoEPG will further add to the experimental tractability of the C. elegans pharynx as a model neural circuit.     

Individual and Population Level Effects of Partner Notification for Chlamydia trachomatis

Partner notification (PN or contact tracing) is an important aspect of treating bacterial sexually transmitted infections (STIs), such as Chlamydia trachomatis. It facilitates the identification of new infected cases that can be treated through individual case management. PN also acts indirectly by limiting onward transmission in the general population. However, the impact of PN, both at the level of individuals and the population, remains unclear. Since it is difficult to study the effects of PN empirically, mathematical and computational models are useful tools for investigating its potential as a public health intervention. To this end, we developed an individual-based modeling framework called Rstisim. It allows the implementation of different models of STI transmission with various levels of complexity and the reconstruction of the complete dynamic sexual partnership network over any time period. A key feature of this framework is that we can trace an individual’s partnership history in detail and investigate the outcome of different PN strategies for C. trachomatis. For individual case management, the results suggest that notifying three or more partners from the preceding 18 months yields substantial numbers of new cases. In contrast, the successful treatment of current partners is most important for preventing re-infection of index cases and reducing further transmission of C. trachomatis at the population level. The findings of this study demonstrate the difference between individual and population level outcomes of public health interventions for STIs.     

The Role of Platelet Serotonin and Depression in the Acute Coronary Syndrome Population

Platelet serotonin has been associated with depression and coronary artery disease. Understanding the association between platelet serotonin and depressive symptoms during acute coronary syndrome (ACS) may explain some of the ACS events seen in depressed individuals. The objectives were to evaluate whether levels of platelet serotonin during an ACS event differ between individuals who screen positive or negative for depressive symptoms and to determine if a linear relationship exists. In this cross-sectional study, data were collected on 51 patients with ACS. Multiple linear regression models were examined. Platelet serotonin levels were not significantly different between the depressed and non-depressed groups (β = -4.093 and p = .293); a linear relationship was not found (β = -.254 and p = .250). In conclusion, a relationship between platelet serotonin and depressive symptoms was not found. It remains unclear if an association exists between platelet serotonin levels and depressive symptoms during hospitalization for ACS.     

Exposure to Mitochondrial Genotoxins and Dopaminergic Neurodegeneration in Caenorhabditis elegans

Neurodegeneration has been correlated with mitochondrial DNA (mtDNA) damage and exposure to environmental toxins, but causation is unclear. We investigated the ability of several known environmental genotoxins and neurotoxins to cause mtDNA damage, mtDNA depletion, and neurodegeneration in Caenorhabditis elegans. We found that paraquat, cadmium chloride and aflatoxin B₁ caused more mitochondrial than nuclear DNA damage, and paraquat and aflatoxin B₁ also caused dopaminergic neurodegeneration. 6-hydroxydopamine (6-OHDA) caused similar levels of mitochondrial and nuclear DNA damage. To further test whether the neurodegeneration could be attributed to the observed mtDNA damage, C. elegans were exposed to repeated low-dose ultraviolet C radiation (UVC) that resulted in persistent mtDNA damage; this exposure also resulted in dopaminergic neurodegeneration. Damage to GABAergic neurons and pharyngeal muscle cells was not detected. We also found that fasting at the first larval stage was protective in dopaminergic neurons against 6-OHDA-induced neurodegeneration. Finally, we found that dopaminergic neurons in C. elegans are capable of regeneration after laser surgery. Our findings are consistent with a causal role for mitochondrial DNA damage in neurodegeneration, but also support non mtDNA-mediated mechanisms.     

Characterization of N-Acyl Phosphatidylethanolamine-Specific Phospholipase-D Isoforms in the Nematode Caenorhabditis elegans

N-acylethanolamines are an important class of lipid signaling molecules found in many species, including the nematode Caenorhabditis elegans (C. elegans) where they are involved in development and adult lifespan. In mammals, the relative activity of the biosynthetic enzyme N-acyl phosphatidylethanolamine-specific phospholipase-D and the hydrolytic enzyme fatty acid amide hydrolase determine N-acylethanolamine levels. C. elegans has two N-acyl phosphatidylethanolamine-specific phospholipase-D orthologs, nape-1 and nape-2, that are likely to have arisen from a gene duplication event. Here, we find that recombinant C. elegans NAPE-1 and NAPE-2 are capable of generating N-acylethanolamines in vitro, confirming their functional conservation. In vivo, they exhibit overlapping expression in the pharynx and the nervous system, but are also expressed discretely in these and other tissues, suggesting divergent roles. Indeed, nape-1 over-expression results in delayed growth and shortened lifespan only at 25°C, while nape-2 over-expression results in significant larval arrest and increased adult lifespan at 15°C. Interestingly, deletion of the N-acylethanolamine degradation enzyme faah-1 exacerbates nape-1 over-expression phenotypes, but suppresses the larval arrest phenotype of nape-2 over-expression, suggesting that faah-1 is coupled to nape-2, but not nape-1, in a negative feedback loop. We also find that over-expression of either nape-1 or nape-2 significantly enhances recovery from the dauer larval stage in the insulin signaling mutant daf-2(e1368), but only nape-1 over-expression reduces daf-2 adult lifespan, consistent with increased levels of the N-acylethanolamine eicosapentaenoyl ethanolamine. These results provide evidence that N-acylethanolamine biosynthetic enzymes in C. elegans have conserved function and suggest a temperature-dependent, functional divergence between the two isoforms.     

Modulation of Locomotion and Reproduction by FLP Neuropeptides in the Nematode Caenorhabditis elegans

Neuropeptides function in animals to modulate most, if not all, complex behaviors. In invertebrates, neuropeptides can function as the primary neurotransmitter of a neuron, but more generally they co-localize with a small molecule neurotransmitter, as is commonly seen in vertebrates. Because a single neuron can express multiple neuropeptides and because neuropeptides can bind to multiple G protein-coupled receptors, neuropeptide actions increase the complexity by which the neural connectome can be activated or inhibited. Humans are estimated to have 90 plus neuropeptide genes; by contrast, nematodes, a relatively simple organism, have a slightly larger complement of neuropeptide genes. For instance, the nematode Caenorhabditis elegans has over 100 neuropeptide-encoding genes, of which at least 31 genes encode peptides of the FMRFamide family. To understand the function of this large FMRFamide peptide family, we isolated knockouts of different FMRFamide-encoding genes and generated transgenic animals in which the peptides are overexpressed. We assayed these animals on two basic behaviors: locomotion and reproduction. Modulating levels of different neuropeptides have strong as well as subtle effects on these behaviors. These data suggest that neuropeptides play critical roles in C. elegans to fine tune neural circuits controlling locomotion and reproduction.     

Functional Genomic Analysis of the let-7 Regulatory Network in Caenorhabditis elegans

The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7–dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development.     

Variation,Sex, and Social Cooperation: Molecular Population Genetics of the Social Amoeba Dictyostelium discoideum

Dictyostelium discoideum is a eukaryotic microbial model system for multicellular development, cell–cell signaling, and social behavior. Key models of social evolution require an understanding of genetic relationships between individuals across the genome or possibly at specific genes, but the nature of variation within D. discoideum is largely unknown. We re-sequenced 137 gene fragments in wild North American strains of D. discoideum and examined the levels and patterns of nucleotide variation in this social microbial species. We observe surprisingly low levels of nucleotide variation in D. discoideum across these strains, with a mean nucleotide diversity (π) of 0.08%, and no strong population stratification among North American strains. We also do not find any clear relationship between nucleotide divergence between strains and levels of social dominance and kin discrimination. Kin discrimination experiments, however, show that strains collected from the same location show greater ability to distinguish self from non-self than do strains from different geographic areas. This suggests that a greater ability to recognize self versus non-self may arise among strains that are more likely to encounter each other in nature, which would lead to preferential formation of fruiting bodies with clonemates and may prevent the evolution of cheating behaviors within D. discoideum populations. Finally, despite the fact that sex has rarely been observed in this species, we document a rapid decay of linkage disequilibrium between SNPs, the presence of recombinant genotypes among natural strains, and high estimates of the population recombination parameter ρ. The SNP data indicate that recombination is widespread within D. discoideum and that sex as a form of social interaction is likely to be an important aspect of the life cycle.     

Intracellular Trafficking and Synaptic Function of APL-1 in Caenorhabditis elegans

Background

Alzheimer''s disease (AD) is a neurodegenerative disorder primarily characterized by the deposition of β-amyloid plaques in the brain. Plaques are composed of the amyloid-β peptide derived from cleavage of the amyloid precursor protein (APP). Mutations in APP lead to the development of Familial Alzheimer''s Disease (FAD), however, the normal function of this protein has proven elusive. The organism Caenorhabditis elegans is an attractive model as the amyloid precursor-like protein (APL-1) is the single ortholog of APP, and loss of apl-1 leads to a severe molting defect and early larval lethality.

Methodology/Principal Findings

We report here that lethality and molting can be rescued by full length APL-1, C-terminal mutations as well as a C-terminal truncation, suggesting that the extracellular region of the protein is essential for viability. RNAi knock-down of apl-1 followed by drug testing on the acetylcholinesterase inhibitor aldicarb showed that loss of apl-1 leads to aldicarb hypersensitivity, indicating a defect in synaptic function. The aldicarb hypersensitivity can be rescued by full length APL-1 in a dose dependent fashion. At the cellular level, kinesins UNC-104/KIF-1A and UNC-116/kinesin-1 are positive regulators of APL-1 expression in the neurons. Knock-down of the small GTPase rab-5 also leads to a dramatic decrease in the amount of apl-1 expression in neurons, suggesting that trafficking from the plasma membrane to the early endosome is important for apl-1 function. Loss of function of a different small GTPase, UNC-108, on the contrary, leads to the retention of APL-1 in the cell body.

Conclusions/Significance

Our results reveal novel insights into the intracellular trafficking of APL-1 and we report a functional role for APL-1 in synaptic transmission.     

Individual differences in young rhesus monkeys: Consistency and change

At 1 year and 2.5 years of age, rhesus monkeys were removed from the colony to a strange situation. Upon introduction, each monkey was watched for 2 hr, given a series of behavioural tests, and watched again six days after introduction. Activity increased with age, as did readiness to approach a ball. Time spent looking at a mirror decreased with age. Although test measures were reliable at each age, there was no significantly positive correlation from one age to the next. Nevertheless, with males, but not females, significant correlations occurred at each age between behaviour in the strange situation and observers’ ratings of behaviour in the colony.     

Bacterial Fatty Acids Enhance Recovery from the Dauer Larva in Caenorhabditis elegans

The dauer larva is a specialized dispersal stage in the nematode Caenorhabditis elegans that allows the animal to survive starvation for an extended period of time. The dauer does not feed, but uses chemosensation to identify new food sources and to determine whether to resume reproductive growth. Bacteria produce food signals that promote recovery of the dauer larva, but the chemical identities of these signals remain poorly defined. We find that bacterial fatty acids in the environment augment recovery from the dauer stage under permissive conditions. The effect of increased fatty acids on different dauer constitutive mutants indicates a role for insulin peptide secretion in coordinating recovery from the dauer stage in response to fatty acids. These data suggest that worms can sense the presence of fatty acids in the environment and that elevated levels can promote recovery from dauer arrest. This may be important in the natural environment where the dauer larva needs to determine whether the environment is appropriate to support reproductive growth following dauer exit.