Coordinated rhythmic movements are ubiquitous in animal behavior. In many organisms, chains of neural oscillators underlie the generation of these rhythms. In C. elegans, locomotor wave generation has been poorly understood; in particular, it is unclear where in the circuit rhythms are generated, and whether there exists more than one such generator. We used optogenetic and ablation experiments to probe the nature of rhythm generation in the locomotor circuit. We found that multiple sections of forward locomotor circuitry are capable of independently generating rhythms. By perturbing different components of the motor circuit, we localize the source of secondary rhythms to cholinergic motor neurons in the midbody. Using rhythmic optogenetic perturbation, we demonstrate bidirectional entrainment of oscillations between different body regions. These results show that, as in many other vertebrates and invertebrates, the C. elegans motor circuit contains multiple oscillators that coordinate activity to generate behavior.

Ca2+ is a key signal transducer for muscle contraction. Continuous in vivo monitoring of intracellular Ca2+-dynamics in C. elegans pharynx muscle revealed surprisingly complex Ca2+ patterns. Despite the age-dependent decline of pharynx pumping, we observed unaltered fast Ca2+ oscillations both in young and old worms. In addition, sporadic prolonged Ca2+ increases lasting many seconds or minutes were often observed in between periods of fast Ca2+ oscillations. We attribute them to the inhibition of ATP-dependent Ca2+-pumps upon energy depletion. Accordingly, food deprivation largely augmented the frequency of prolonged [Ca2+] increases. However, paradoxically, prolonged [Ca2+] increases were more frequently observed in young worms than in older ones, and less frequently observed in energy-deficient mitochondrial respiratory chain nuo-6 mutants than in wild-type controls. We hypothesize that young animals are more susceptible to energy depletion due to their faster energy consumption rate, while nuo-6 mutants may keep better the energy balance by slowing energy consumption. Our data therefore suggest that the metabolic state of the pharynx during feeding stimulation depends mainly on the delicate balance between the instant rates of energy production and consumption. Thus, in vivo monitoring of muscle Ca2+ dynamics can be used as a novel tool to study cellular energy availability.

MICU1 and MICU2 are the main regulators of the mitochondrial Ca(2+)-uniporter (MCU), but their precise functional role is still under debate. We show here that MICU2 behaves as a pure inhibitor of MCU at low cytosolic [Ca(2+)] ([Ca(2+)]c), though its effects decrease as [Ca(2+)]c is increased and disappear above 7 μM. Regarding MICU1, studying its effects is more difficult because knockdown of MICU1 leads also to loss of MICU2. However, while knockdown of MICU2 induces only a persistent increase in mitochondrial Ca(2+) uptake, knockdown of MICU1 also induces a peculiar use-dependent transient activation of MCU that cannot be attributed to the parallel loss of MICU2. Therefore, MICU1 is endowed with a specific inhibitory effect on MCU at low [Ca(2+)]c, separate and kinetically different from that of MICU2. On the other hand, we and others have shown previously that MICU1 activates MCU at [Ca(2+)]c above 2.5 μM. Thus, MICU1 has a double role in MCU regulation, inhibitory at low [Ca(2+)]c and activatory at high [Ca(2+)]c.

Ca2+ is a ubiquitous second messenger that plays an essential role in physiological processes such as muscle contraction, neuronal secretion, and cell proliferation or differentiation. There is ample evidence that the dysregulation of Ca2+ signaling is one of the key events in the development of neurodegenerative processes, an idea called the “calcium hypothesis” of neurodegeneration. Caenorhabditis elegans (C. elegans) is a very good model for the study of aging and neurodegeneration. In fact, many of the signaling pathways involved in longevity were first discovered in this nematode, and many models of neurodegenerative diseases have also been developed therein, either through mutations in the worm genome or by expressing human proteins involved in neurodegeneration (β-amyloid, α-synuclein, polyglutamine, or others) in defined worm tissues. The worm is completely transparent throughout its whole life, which makes it possible to carry out Ca2+ dynamics studies in vivo at any time, by expressing Ca2+ fluorescent probes in defined worm tissues, and even in specific organelles such as mitochondria. This review will summarize the evidence obtained using this model organism to understand the role of Ca2+ signaling in aging and neurodegeneration.

Progressive decline in mitochondrial function is generally considered one of the hallmarks of aging. We have expressed a Ca2+ sensor in the mitochondrial matrix of C. elegans pharynx cells and we have measured for the first time mitochondrial [Ca2+] ([Ca2+]M) dynamics in the pharynx of live C. elegans worms during aging. Our results show that worms stimulated with serotonin display a pharynx [Ca2+]M oscillatory kinetics that includes both high frequency oscillations (up to about 1Hz) and very prolonged “square-wave” [Ca2+]M increases, indicative of energy depletion of the pharynx cells. Mitochondrial [Ca2+] is therefore able to follow “beat-to-beat” the fast oscillations of cytosolic [Ca2+]. The fast [Ca2+]M oscillations kept steady frequency values during the whole worm life, from 2 to 12 days old, but the height and width of the peaks was progressively reduced. [Ca2+]M oscillations were also present with similar kinetics in respiratory chain complex I nuo-6 mutant worms, although with smaller height and frequency than in the controls, and larger width. In summary, Ca2+ fluxes in and out of the mitochondria are relatively well preserved during the C. elegans life, but there is a clear progressive decrease in their magnitude during aging. Moreover, mitochondrial Ca2+ fluxes were smaller in nuo-6 mutants with respect to the controls at every age and decreased similarly during aging.

Neural circuits coordinate with muscles and sensory feedback to generate motor behaviors appropriate to an animal’s environment. In C. elegans, the mechanisms by which the motor circuit generates undulations and modulates them based on the environment are largely unclear. We quantitatively analyzed C. elegans locomotion during free movement and during transient optogenetic muscle inhibition. Undulatory movements were highly asymmetrical with respect to the duration of bending and unbending during each cycle. Phase response curves induced by brief optogenetic inhibition of head muscles showed gradual increases and rapid decreases as a function of phase at which the perturbation was applied. A relaxation oscillator model based on proprioceptive thresholds that switch the active muscle moment was developed and is shown to quantitatively agree with data from free movement, phase responses, and previous results for gait adaptation to mechanical loadings. Our results suggest a neuromuscular mechanism underlying C. elegans motor pattern generation within a compact circuit.

The sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) refills the endoplasmic reticulum (ER) with Ca2+ up to the millimolar range and is therefore the main controller of the ER [Ca2+] level ([Ca2+]ER), which has a key role in the modulation of cytosolic Ca2+ signaling and ER-mitochondria Ca2+ transfer. Given that both cytosolic and mitochondrial Ca2+ dynamics strongly interplay with energy metabolism and nutrient-sensitive pathways, both of them involved in the aging process, we have studied the effect of SERCA inhibitors on lifespan in C. elegans. We have used thapsigargin and 2,5-Di-tert-butylhydroquinone (2,5-BHQ) as SERCA inhibitors, and the inactive analog 2,6-Di-tert-butylhydroquinone (2,6-BHQ) as a control for 2,5-BHQ. Every drug was administered to the worms either directly in the agar or via an inclusion compound with γ-cyclodextrin. The results show that 2,6-BHQ produced a small but significant increase in survival, perhaps because of its antioxidant properties. However, 2,5-BHQ produced in all the conditions a much higher increase in lifespan, and the potent and specific SERCA inhibitor thapsigargin also extended the lifespan. The effects of 2,5-BHQ and thapsigargin had a bell-shaped concentration dependence, with a maximum effect at a certain dose and smaller or even toxic effects at higher concentrations. Our data show therefore that submaximal inhibition of SERCA pumps has a pro-longevity effect, suggesting that Ca2+ signaling plays an important role in the aging process and that it could be a promising novel target pathway to act on aging.

Alzheimer’s disease (AD) is the most frequent cause of dementia. After decades of research, we know the importance of the accumulation of protein aggregates such as β-amyloid peptide and phosphorylated tau. We also know that mutations in certain proteins generate early-onset Alzheimer’s disease (EOAD), and many other genes modulate the disease in its sporadic form. However, the precise molecular mechanisms underlying AD pathology are still unclear. Because of ethical limitations, we need to use animal models to investigate these processes. The nematode Caenorhabditis elegans has received considerable attention in the last 25 years, since the first AD models overexpressing Aβ peptide were described. We review here the main results obtained using this model to study AD. We include works studying the basic molecular mechanisms of the disease, as well as those searching for new therapeutic targets. Although this model also has important limitations, the ability of this nematode to generate knock-out or overexpression models of any gene, single or combined, and to carry out toxicity, recovery or survival studies in short timeframes with many individuals and at low cost is difficult to overcome. We can predict that its use as a model for various diseases will certainly continue to increase.