Ratiometric Calcium Imaging of Individual Neurons in Behaving &lt;em&gt;Caenorhabditis Elegans&lt;/em&gt;

Ravi, Bhavya; Nassar, Layla M.; Kopchock, Richard J.; Dhakal, Pravat; Scheetz, M.; Collins, Kevin M.

doi:10.3791/56911-v

Cited by 2 publications

(4 citation statements)

References 11 publications

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“…The uv1 cells sit at the dorsa-lateral surface of the vulval canal, adjacent to the HSN and VC presynaptic termini that release neurotransmitters onto the vm2 vulval muscle arms (Collins and Koelle, 2013; Ravi et al, 2018a). We developed ratiometric Ca 2+ imaging methods to record in vivo cell activity in the circuit by using the calcium reporter GCaMP5 co-expressed with the Ca 2+- insensitive red fluorescent protein mCherry (Ravi et al, 2018b). Our previous uv1 Ca 2+ reporter based on the ocr-2 promoter/enhancer expressed rather weakly in the uv1 cells and with strong additional co-expression in head and tail sensory neurons along with the uterine seam (utse) cells (Collins et al, 2016; Jose et al, 2007).…”

Section: Resultsmentioning

confidence: 99%

“…Ratiometric Ca 2+ recordings were then imported into Volocity (Version 6.5.1, Quorum Technologies, Inc.) for subsequent GCaMP5/mCherry ratiometric analysis. Ca 2+ quantitation of GCaMP5/mCherry ratio changes (ΔR/R), Ca 2+ transient peaks were analyzed through MATLAB as described (Collins et al, 2016; Ravi et al, 2018b).…”

Section: Methodsmentioning

confidence: 99%

“…Ratiometric Ca 2+ imaging of uv1 activity of alive worms were performed as described (Ravi et al, 2018b). GCaMP5 and mCherry fluorescence was excited at 470 nm and 590 nm, respectively, using a Colibri.2 LED illumination system and channels were separated by a Hamamatsu W-View Gemini beam splitter.…”

Section: Methodsmentioning

confidence: 99%

“…Late L4 worms were staged onto plates with or without ATR 24 h prior to the experiment. Ratiometric Ca 2+ imaging in freely behaving animals was performed as described previously (Ravi et al, 2018b) with the same 470 nm blue light used to both activate ChR2 and excite GCaMP5 fluorescence. The brightfield and fluorescence image acquisition was synchronized at 20 fps, and each recording lasted 2-3 mins.…”

Section: Optogeneticsmentioning

confidence: 99%

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TheC. elegansuv1 neuroendocrine cells provide direct mechanosensory feedback of vulval opening

Yan,

Claman,

Bode

et al. 2024

Preprint

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Neuroendocrine cells react to physical, chemical, and synaptic signals originating from tissues and the nervous system, releasing hormones that regulate various body functions beyond the synapse. Neuroendocrine cells are often embedded in complex tissues making direct tests of their activation mechanisms and signaling effects difficult to study. In the nematode worm C. elegans, four uterine-vulval (uv1) neuroendocrine cells sit above the vulval canal next to the egg-laying circuit, releasing tyramine and neuropeptides that feedback to inhibit egg laying. We have previously shown uv1 cells are mechanically deformed during egg laying, driving uv1 Ca2+ transients. However, whether egg-laying circuit activity, vulval opening, and/or egg release triggered uv1 Ca2+ activity was unclear. Here we show uv1 responds directly to mechanical activation. Optogenetic vulval muscle stimulation triggers uv1 Ca2+ activity following muscle contraction even in sterile animals. Direct mechanical prodding with a glass probe placed against the worm cuticle triggers robust uv1 Ca2+ activity similar to that seen during egg laying. Direct mechanical activation of uv1 cells does not require other cells in the egg-laying circuit, synaptic or peptidergic neurotransmission, or TRPV and Piezo channels. EGL-19 L-type Ca2+ channels, but not P/Q/N-type or Ryanodine Receptor Ca2+ channels, promote uv1 Ca2+ activity following mechanical activation. L-type channels also facilitate the coordinated activation of uv1 cells across the vulva, suggesting mechanical stimulation of one uv1 cells cross-activates the other. Our findings show how neuroendocrine cells like uv1 report on the mechanics of tissue deformation and muscle contraction, facilitating feedback to local circuits to coordinate behavior.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Optogeneticsmentioning

confidence: 99%

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TheC. elegansuv1 neuroendocrine cells provide direct mechanosensory feedback of vulval opening

Yan,

Claman,

Bode

et al. 2024

Preprint

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Serotonin signals through postsynaptic Gα_q, Trio RhoGEF, and diacylglycerol to promoteC. elegansegg-laying circuit activity and behavior

Dhakal

Chaudhry

Collins

2021

Preprint

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Activated Gαq signals through Phospholipase-Cβ (PLCβ) and Trio, a Rho GTPase exchange factor (RhoGEF), but how these two effector pathways promote synaptic transmission remains poorly understood. We used the egg-laying behavior circuit of C. elegans to determine whether PLCβ and Trio mediate serotonin and Gαq signaling through independent or related biochemical pathways. Using genetic rescue experiments, we find that PLCβ functions in neurons while Trio functions in both neurons and the postsynaptic vulval muscles. While Gαq, PLCβ, and Trio RhoGEF mutants all fail to lay eggs in response to serotonin, optogenetic stimulation of the serotonin releasing HSN command neurons restores egg laying only in PLCβ mutants. Vulval muscle Ca2+ activity remained in PLCβ mutants but was eliminated in strong Gαq and Trio RhoGEF mutants. Exogenous treatment with Phorbol esters that mimic Diacylglycerol (DAG), a product of PIP2 hydrolysis, rescued egg-laying circuit activity and behavior defects of Gαq signaling mutants, suggesting both Phospholipase C and Rho signaling promote synaptic transmission and egg-laying via DAG production. DAG has been proposed to activate effectors including UNC-13, however, we find that phorbol esters, but not serotonin, stimulate egg laying in unc-13 mutants. Together, these results show that serotonin signaling through Gαq and PLCβ modulates UNC-13 activity to promote neurotransmitter release. Serotonin also signals through Gαq, Trio RhoGEF, and an unidentified PMA-responsive effector to promote postsynaptic muscle excitability. Thus, the same neuromodulator serotonin can signal in distinct cells and effector pathways to activate a motor behavior circuit.

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Ratiometric Calcium Imaging of Individual Neurons in Behaving <em>Caenorhabditis Elegans</em>

Cited by 2 publications

References 11 publications

TheC. elegansuv1 neuroendocrine cells provide direct mechanosensory feedback of vulval opening

TheC. elegansuv1 neuroendocrine cells provide direct mechanosensory feedback of vulval opening

Serotonin signals through postsynaptic Gα_q, Trio RhoGEF, and diacylglycerol to promoteC. elegansegg-laying circuit activity and behavior

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Ratiometric Calcium Imaging of Individual Neurons in Behaving <em>Caenorhabditis Elegans</em>

Cited by 2 publications

References 11 publications

TheC. elegansuv1 neuroendocrine cells provide direct mechanosensory feedback of vulval opening

TheC. elegansuv1 neuroendocrine cells provide direct mechanosensory feedback of vulval opening

Serotonin signals through postsynaptic Gαq, Trio RhoGEF, and diacylglycerol to promoteC. elegansegg-laying circuit activity and behavior

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Serotonin signals through postsynaptic Gα_q, Trio RhoGEF, and diacylglycerol to promoteC. elegansegg-laying circuit activity and behavior