Visualizing calcium flux in freely moving nematode embryos

Author Posting. © The Author(s), 2017. This is the author's version of the work. It is posted here by permission of Cell Press for personal use, not for redistribution. The definitive version was published in Biophysical Journal 112 (2017): 1975-1983, doi:10.1016/j.bpj.2017.02.035.The lack of physiological recordings from Caenorhabditis elegans embryos stands in stark contrast to the comprehensive anatomical and gene expression datasets already available. Using light-sheet fluorescence microscopy (LSFM) to address the challenges associated with functional imaging at this developmental stage, we recorded calcium dynamics in muscles and neurons and developed analysis strategies to relate activity and movement. In muscles, we found that the initiation of twitching was associated with a spreading calcium wave in a dorsal muscle bundle. Correlated activity in muscle bundles was linked with early twitching and eventual coordinated movement. To identify neuronal correlates of behavior, we monitored brain-wide activity with subcellular resolution and identified a particularly active cell associated with muscle contractions. Finally, imaging neurons of a well-defined adult motor circuit, we found that reversals in the eggshell correlated with calcium transients in AVA interneurons.E.A. and A.K. acknowledge support from the Grass Fellowship Program and D. C-R. and H.S. acknowledge the Whitman Fellowship program at MBL. This work was supported by the intramural research program of the National Institute of Biomedical Imaging and Bioengineering and NIH grants U01 HD075602 and R24OD016474 to D.C-R and A.K.2018-05-0

Reflective imaging improves spatiotemporal resolution and collection efficiency in light sheet microscopy

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Nature Communications 8 (2017): 1452, doi:10.1038/s41467-017-01250-8.Light-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, and gentle imaging of live specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of four complementary views in 250 ms, doubling speed and improving information content relative to symmetric dual-view LSFM. We also report a modified deconvolution algorithm that removes associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to <300 nm in all three dimensions) by applying our method to single-view LSFM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture. We demonstrate the broad applicability of our method in a variety of samples, studying mitochondrial, membrane, Golgi, and microtubule dynamics in cells and calcium activity in nematode embryos.This work was supported by the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering at the National Institutes of Health. P.L. and H.S. acknowledge summer support from the Marine Biological Laboratory at Woods Hole, through the Whitman- and Fellows- program. P.L. acknowledges support from NIH National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health (NIH) under grant number R01EB017293. C.S. acknowledges funding from the National Institute of General Medical Sciences of NIH under Award Number R25GM109439 (Project Title: University of Chicago Initiative for Maximizing Student Development [IMSD]) and NIBIB under grant number T32 EB002103. Partial funding for the computation in this work was provided by NIH grant numbers S10 RRO21039 and P30 CA14599. A.U. and I.R.-S. were supported by the NSF grant number 1607645

EFHC1, Implicated in Juvenile Myoclonic Epilepsy, Functions at the Cilium and Synapse to Modulate Dopamine Signaling

Neurons throughout the mammalian brain possess non-motile cilia, organelles with varied functions in sensory physiology and cellular signaling. Yet, the roles of cilia in these neurons are poorly understood. To shed light into their functions, we studied EFHC1, an evolutionarily conserved protein required for motile cilia function and linked to a common form of inherited epilepsy in humans, juvenile myoclonic epilepsy (JME). We demonstrate that C. elegans EFHC-1 functions within specialized non-motile mechanosensory cilia, where it regulates neuronal activation and dopamine signaling. EFHC-1 also localizes at the synapse, where it further modulates dopamine signaling in cooperation with the orthologue of an R-type voltage-gated calcium channel. Our findings unveil a previously undescribed dual-regulation of neuronal excitability at sites of neuronal sensory input (cilium) and neuronal output (synapse). Such a distributed regulatory mechanism may be essential for establishing neuronal activation thresholds under physiological conditions, and when impaired, may represent a novel pathomechanism for epilepsy

Data from: Insights into the roles of CMK-1 and OGT-1 in interstimulus interval-dependent habituation in Caenorhabditis elegans

Habituation is a ubiquitous form of non-associative learning observed as a decrement in responding to repeated stimulation that cannot be explained by sensory adaptation or motor fatigue. One of the defining characteristics of habituation is its sensitivity to the rate at which training stimuli are presented – animals habituate faster in response to more rapid stimulation. The molecular mechanisms underlying this interstimulus interval (ISI)-dependent characteristic of habituation remain unknown. In this article we use behavioral neurogenetic and bioinformatic analyses in the nematode Caenorhabiditis elegans to identify the first molecules that modulate habituation in an ISI-dependent manner. We show that the Caenorhabditis elegans orthologs of Ca2+/calmodulin-dependent kinases CaMK1/4, CMK-1, and O-linked N-acetylglucosamine (O-GlcNAc) transferase, OGT-1, both function in primary sensory neurons to inhibit habituation at short ISIs and promote it at long ISIs. In addition, both cmk-1 and ogt-1 mutants display a rare mechanosensory hyper-responsive phenotype, i.e. larger mechanosensory responses than wild-type. Overall, our work identifies two conserved genes that function in sensory neurons to modulate habituation in an ISI-dependent manner, providing the first insights into the molecular mechanisms underlying the universally observed phenomenon that habituation has different properties when stimuli are delivered at different rates

An afferent neuropeptide system transmits mechanosensory signals triggering sensitization and arousal in C. elegans

Sensitization is a simple form of behavioral plasticity by which an initial stimulus, often signaling danger, leads to increased responsiveness to subsequent stimuli. Cross-modal sensitization is an important feature of arousal in many organisms, yet its molecular and neural mechanisms are incompletely understood. Here we show that in C. elegans, aversive mechanical stimuli lead to both enhanced locomotor activity and sensitization of aversive chemosensory pathways. Both locomotor arousal and cross-modal sensitization depend on the release of FLP-20 neuropeptides from primary mechanosensory neurons and on their receptor FRPR-3. Surprisingly, the critical site of action of FRPR-3 for both sensory and locomotor arousal is RID, a single neuroendocrine cell specialized for the release of neuropeptides that responds to mechanical stimuli in a FLP-20-dependent manner. Thus, FLP-20 peptides function as an afferent arousal signal that conveys mechanosensory information to central neurons that modulate arousal and other behavioral states.status: publishe

Data from: EFHC1, implicated in juvenile myoclonic epilepsy, functions at the cilium and synapse to modulate dopamine signaling

Neurons throughout the mammalian brain possess non-motile cilia, organelles with varied functions in sensory physiology and cellular signaling, yet their roles in these neurons are poorly understood. To shed light into their functions, we studied EFHC1, an evolutionarily conserved protein required for motile cilia function and linked to a common form of inherited epilepsy in humans, juvenile myoclonic epilepsy (JME). We demonstrate that C. elegans EFHC1 functions within specialized non-motile mechanosensory cilia, where it regulates neuronal activation and dopamine signaling. EFHC1 also localizes at the synapse, where it further modulates dopamine signaling in cooperation with the orthologue of an R-type voltage-gated calcium channel. Our findings unveil a previously undescribed dual-regulation of neuronal excitability at sites of neuronal sensory input (cilium) and neuronal output (synapse). Such a distributed regulatory mechanism may be essential for establishing neuronal activation thresholds under physiological conditions, and when impaired, may represent a novel pathomechanism for epilepsy