An hourglass circuit motif transforms a motor program via subcellularly localized muscle calcium signaling and contraction
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Abstract
Neural control of muscle function is fundamental to animal behavior. Many muscles can generate multiple distinct behaviors. Nonetheless, individual muscle cells are generally regarded as the smallest units of motor control. We report that muscle cells can alter behavior by contracting subcellularly. We previously discovered that noxious tastes reverse the net flow of particles through the C. elegans pharynx, a neuromuscular pump, resulting in spitting. We now show that spitting results from the subcellular contraction of the anterior region of the pm3 muscle cell. Subcellularly localized calcium increases accompany this contraction. Spitting is controlled by an ‘hourglass’ circuit motif: parallel neural pathways converge onto a single motor neuron that differentially controls multiple muscles and the critical subcellular muscle compartment. We conclude that subcellular muscle units enable modulatory motor control and propose that subcellular muscle contraction is a fundamental mechanism by which neurons can reshape behavior.
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on July 6 2020, follows.
Summary
Sando et al. extend on previous work by the same lab to delineate the neuronal mechanisms that control UV-light / ROS suppression of feeding and evoked spitting behaviors. They provide a nice characterization of pharyngeal behaviors that are involved in feeding and spitting, showing that upon UV-light stimulation feeding pumps are modulated to evoke spitting instead. M1 neurons are central to the spitting reflex; they sense light, integrate inputs from light sensitive I2 and I4 neurons and transmit the information to the anterior pharyngeal muscles pm1/2 and the anterior part of pm3. The conceptual advances of this paper are twofold:
The hourglass circuit motif as a means to transform ingestion movements …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on July 6 2020, follows.
Summary
Sando et al. extend on previous work by the same lab to delineate the neuronal mechanisms that control UV-light / ROS suppression of feeding and evoked spitting behaviors. They provide a nice characterization of pharyngeal behaviors that are involved in feeding and spitting, showing that upon UV-light stimulation feeding pumps are modulated to evoke spitting instead. M1 neurons are central to the spitting reflex; they sense light, integrate inputs from light sensitive I2 and I4 neurons and transmit the information to the anterior pharyngeal muscles pm1/2 and the anterior part of pm3. The conceptual advances of this paper are twofold:
The hourglass circuit motif as a means to transform ingestion movements into spits.
Local activation of pm3 muscles via a compartmentalized calcium signal that ensures opening of only the anterior part of the alimentary tract.
Most of the behavioral experiments are well done and the paper could be of potential interest to a broad audience. However, the reviewers raised some concerns that should be addressed prior to publication in eLife.
Essential Revisions
- A major concern is that all three reviewers are not convinced that the data presented here support the conclusion of local calcium dynamics in the anterior pm3 muscles. Since this is one of the major aspects of this study, it is essential to provide more experimental evidence. The authors used a pan-pharyngeal driver to express GCaMP. The imaging resolution seems not good enough to distinguish calcium transients in pm1/2/3 and the most straight forward interpretation of the results is that the anterior calcium transients are derived from pm1/2 but not pm3. It seems otherwise to rest on the claim that pm3 is sufficient for spitting and that, in the absence of pm1/2, local contraction of pm3 is the only way to hold the valve open during expulsion. Same for Fig 4F.
To substantiate the claim, these experiments should be repeated using a pm3 specific driver.
Alternatively, if pm3 specific drivers are not available, the experiments could be repeated upon laser ablation of pm1/2, to ensure that the signals are indeed specifically derived from pm3.
Perhaps, if imaging resolution and interference by emission light scattering permits, an overlay of a good DIC with GCaMP fluorescence may settle this more easily since pm3 stops at the base of the buccal cavity whereas pm1/2 line the cavity.
Individual recording traces of the different regions along with ethograms of the pharyngeal behaviors should be shown.
- The authors use a calcium imaging assay in immobilized worms to record UV-light evoked muscle activity- and pharyngeal neuron activity. While pumping and spitting behaviors occur at a frequency of up to 5Hz in the behavioral assays (e.g. Fig 1D,E), calcium dynamics in muscle and neurons were observed at 1-2 orders of magnitude slower (e.g. Fig 1 H,I; Fig 4H-M). However, the authors state that these dynamics would match well the time-scale at which light evoked pumps are observed. This is confusing. While it is possible that pharyngeal neurons encode the rate of pumping/spitting, muscle activity should correspond to the motor rhythms.
What is the pumping rate under the imaging/immobilization conditions? Do the animals spit? The behaviors under imaging conditions need to be better characterized and documented.
Individual traces should be shown throughout (like Fig 4H), importantly next to ethograms of pharyngeal behaviors.
The image acquisition rate should be stated in the methods? Was this also 2Hz like the flickering rate?
Only with this information at hand it is possible to properly interpret the imaging results. Are the measurements convoluted by low acquisition rate and slow on/off kinetics of GCaMP, or do light evoked pharyngeal behaviors occur at such a slow frequency in immobilized worms?
The purported movements of the metastomal filter appear to be based solely on the observation of particle flow with a particular concentration and size of beads. At times this may be misleading. For example, the authors report that 25% of normal pumps are associated with openings of the metastomal filter. However, it is possible that the beads do not always become jammed in the buccal cavity, even if the metastomal flaps remain in position. Direct imaging of the metastomal flaps would address this question; if this is not possible the limitations of the assay should at least be acknowledged.
The opening of the metastomal flaps during spitting is interpreted as a "rinsing" of its mouth "in response to a bad taste". This interpretation is problematic since the animal is "rinsing" its mouth with the same particles that have presumably induced the spitting. It would make more sense if the animal increased rather than decreased selectivity of the metastomal filter; this would allow water to enter the pharynx while excluding potentially toxic particles. If the authors insist in their interpretation they should at least discuss this issue.
Line 183 - What is the basis for believing the sufficiency of pm3 is based on "contraction of a subcellular region"? And Line 188 - where is this "uncoupling" shown? There are few figures/data here. Is it deduced that this must be so because the pharyngeal valve is open while the lumen closes during spitting? Is local contraction of pm3 the only possible explanation for this? In the WT condition, for example, could pm1 and/or pm2 contraction overcome a global relaxation of pm3 to hold the valve upen during lumen closing? Although spitting apparently persists after ablation of pm1/pm2, these events should be documented in the same detail as WT events to demonstrate that pm3 is truly sufficient for "normal" spitting (i.e. continued pumping of lumen while the valve and filter are held open, local Ca++ events in anterior portion of pm3). This section seems to take a leap to a precise muscle mechanism based only on the ablation.
At the cellular level, the authors note that calcium waves in muscle can cause local contraction patterns that lead to peristalsis, but that their observations seem to be of a different kind in terms of spatial and temporal patterning (long sustained local Ca++/contraction in one domain while rhythmic Ca/contraction occur in another domain). How input strength might create such a pattern is difficult to envision, given the simplicity of the M1 pm3 innervation pattern. What is the proposed cellular mechanism here?
Figure 4J-L: these panels lack quantifications. Please show also individual traces; is the little initial bump in lite-1 mutants' response consistent across multiple recordings? Is the reduction in lite-1;gur-3 statistically significant?
Why is this initial transient signal so much stronger when gur-3 is expressed in I2 in the double mutants (Fig 5D)?
Line 422-424: this statement is not supported by data in Fig 6B-F; only I4 ablated animals show a robust defect and there is no synergistic effect in the double ablation.
Fig 6G: this result lacks quantifications. Appropriate statistics should be performed. Show also individual traces.
Line 210 - "data not shown"... the correlation between spatially-restricted contraction / Ca++ signals and spitting is a central claim of the paper...it needs to be quantitatively documented in a figure.
Line 104 - Is the experimenter blinded to strain/condition? If not, what steps were taken to detect or correct experimenter bias? This is a major pitfall of manual behavior coding.
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