Multiple neuronal networks coordinate Hydra mechanosensory behavior
- Department of Bioengineering, Rice University, United States
- Department of Molecular and Cellular Biology, United States
- Department of Electrical and Computer Engineering, Rice University, United States
- Department of Neuroscience, Baylor College of Medicine, United States
Figures
Figure 1
Distribution of neurons in the Hydra nerve net.
(a) Fluorescent image of Hydra nervous system. Green fluorescent protein (GFP) is expressed in neurons and neuronal progenitors (nGreen transgenic line; Siebert et al., 2019). Body anatomy is …
Figure 2 with 9 supplements
Hydra’s neuronal response depends on the mechanical stimulus intensity.
(a) (Left) Side view of double-layer microfluidic device for mechanical stimulation. (Right) Device with pressurized valve. Hydra is immobilized in the bottom Hydra layer, and pressurized air …
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Figure 2—source data 1
source file for stimulus-dependent mechanosensory response.
The file (Fig2_SourceData.mat) contains all source data used for quantitatively characterizing mechanosensory response shown in Figure 2. The file contains a struct named ‘HydraData’ with each trial in a different row. The columns contain the raw and processed data for each trial: ‘StimCondition’ indicates the stimulation pressure (0, 5, 10, 15, 20, or 25 psi) used during the trial; ‘RawFluorescenceFoot’ contains the raw fluorescence values from foot/peduncle region of interest (ROI); ‘StimulationTrace’ contains the trace of stimulus delivery where 0 is valve off, 1 is valve on (and stimulation is being applied) per frame; ‘Response Probability’ is extracted after processing ‘RawFluorescenceFoot’ with stimulation onset times from ‘StimulationTrace’; ‘TimeBetweenContractions’ is time in seconds between contraction burst pulses extracted from ‘RawFluorescenceFoot’; ‘SinglePulseContractions_Percent’ is the percent of body contractions that occur as single pulses extracted from ‘RawFluorescenceFoot’.
- https://cdn.elifesciences.org/articles/64108/elife-64108-fig2-data1-v1.mat
Figure 2—figure supplement 1
Distribution of mechanical forces.
Fluorescent image (grayscale) of transgenic Hydra (nGreen) expressing GFP in neurons and neural progenitors. The oral region is on the left. Aboral//peduncle region is on the right. Arrows (cyan) …
Figure 2—figure supplement 2
Average calcium fluorescence from large peduncle region of interest (ROI) correlated with calcium fluorescence from smaller ROIs for individual peduncle neurons.
(a) Fluorescent image of transgenic Hydra expressing GCaMP6s pan-neuronally. Dashed while circle indicates the peduncle ROI. Small cyan circles indicate the individual neuron ROIs. Peduncle region …
Figure 2—figure supplement 3
Spikes in calcium fluorescence are due to calcium activity not motion artifacts.
(a) Fluorescence image of transgenic Hydra (nGreen) expressing GFP in neurons false colored with hot green colormap. Neurons appear in white. (b) Fluorescence image of transgenic Hydra expressing …
Figure 2—figure supplement 4
Simultaneous electrophysiology and calcium imaging of ectodermal epitheliomuscular cells.
(a) Photograph (left) of a microfluidic immobilization chamber filled with green dye. Black box highlights the recording region in the microfluidic chamber (110 µm tall). False-colored scanning …
Figure 2—figure supplement 5
Hydra’s epitheliomuscular response is dependent on the mechanical stimulus intensity.
(a) Gray trace (top) is the stimulation protocol. 20 min no stimulation, 20 min of repeated stimulation (1 s ‘on,’ 30 s ‘off’) at 15 psi, 20 min of repeated stimulation (1 s ‘on,’ 30 s ‘off’) at 20 …
Figure 2—figure supplement 6
Mechanosensory response window.
(a) Different response windows tested (1 s in blue; 5 s in orange; 10 s in yellow; and 15 s in purple). (Top) The lengths of the rectangles correspond to time (s) in the raster plot below. Stimulus …
Figure 2—figure supplement 7
Mechanical sensitivity of different body regions in Hydra.
(a) Response probability of transgenic Hydra (N = 3 animals expressing GCaMP6s in neurons) stimulated at three different body regions: oral, mid-body, and aboral. Annotated Hydra below the plot …
Figure 2—figure supplement 8
Hydra’s mechanosensory response time is faster than passive calcium diffusion through epitheliomuscular cells.
(a) Distance calcium diffuses passively over given time (left, black line plot) approximated using passive diffusion equation (right). Light brown-shaded region indicates the range of body length …
Figure 2—figure supplement 9
Long-term mechanical stimulation.
(a) Representative calcium fluorescence trace from the peduncle region from an animal (GcaMP6s, neurons) not stimulated and animal mechanically stimulated with 20 psi. Stimulus protocol in gray, 20 …
Figure 3 with 5 supplements
Oral region is important for mechanosensory response.
(a) Representative images of resection preparations (6–12 hr post-resections) of transgenic Hydra during contraction (GCaMP7b, endodermal epitheliomuscular cells): whole (or control), footless, …
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Figure 3—source data 1
Source file of mechanosensory response in resected animals.
The file (Fig3_SourceData.mat) contains all source data used for characterizing mechanosensory response in resected animals in Figure 3. The file contains a struct named ‘HydraData’ with each trial in a different row. The columns contain the raw and processed data for each trial: ‘StimCondition’ indicates stimulation condition (w/or w/o stimulation; body resections – whole, bisected, no foot = footless, no head = headless, no head and foot = BodyColumn); ‘RawFluorescenceFoot’ contains the raw fluorescence values from foot/peduncle region of interest (ROI); ‘StimulationTrace’ contains the trace of stimulus delivery where 0 is valve off, 1 is valve on (and stimulation is being applied) per frame; ‘Response Probability’ is the stimulated contraction probability extracted after processing ‘RawFluorescenceFoot’ with stimulation onset times from ‘StimulationTrace’; ‘BasalContractionProbability’ is spontaneous contraction probability extracted after processing ‘RawFluorescenceFoot’ during (t = 0–20 min) the initial acclimation period without stimulation.
- https://cdn.elifesciences.org/articles/64108/elife-64108-fig3-data1-v1.mat
Figure 3—figure supplement 1
Regeneration of the peduncle network.
(a) Summary schematic of the experiment performed. Dashed line indicates where the incision was made to remove the lower half of the transgenic Hydra body (GcaMP6s, neurons) to discover when the …
Figure 3—figure supplement 2
RT-qPCR analyses of neuron subtype-specific gene expression in resected animals demonstrates loss of specific neuron subtypes.
RT-qPCR was used to test for the loss of specific neuron subtypes in whole, tube (no head or foot), headless, footless, and bisected Hydra using uniquely expressed biomarkers for each subtype. There …
Figure 3—figure supplement 3
Mechanosensory response from endodermal epitheliomuscular cells and neurons in resected Hydra.
(a) Representative images of resection preparations of transgenic Hydra (GCaMP7b, endodermal epitheliomuscular cells): whole (or control), ‘footless,’ ‘headless,’ ‘bisected,’ and ‘body column.’ …
Figure 3—figure supplement 4
Contraction activity in non-stimulated animals.
Cartoon schematic of resection preparations of transgenic Hydra (GCaMP7b, endodermal epitheliomuscular cells): whole (or control), ‘footless,’ ‘headless,’ ‘bisected,’ and ‘body column’ animals. …
Figure 3—figure supplement 5
Hypostome and peduncle nerve rings work together to coordinate contractile behavior.
(a) Representative fluorescence trace used to calculate time interval between contractions. (b) Time interval between spontaneous contractions in animals with different resections: whole, …
Figure 4 with 3 supplements
Distinct networks of neurons involved in spontaneous and stimulated behaviors.
(a) Fluorescent image of transgenic Hydra expressing GCaMP6s pan-neuronally. Individually tracked neurons regions of interest (ROIs) are indicated by arrows, and peduncle ROI is outlined with a …
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Figure 4—source data 1
source file for fluorescent calcium activity from single-neuron regions of interest (ROIs).
The file (Fig4_SourceData.zip) contains three folders ‘Hydra1,’ ‘Hydra2,’ and ‘Hydra3’ for the three different animals with individually tracked single neurons. Each folder contains a file called ‘IndividualNeuronTraces.mat’ with three different matrices: ‘NeuronROI_RawFluorAllFrames’ matrix (# of rows = # of neurons + 1, # of columns = # of frames) contain the raw fluorescence intensities individual neuronal ROIs. Each row is different neuron, and the last row in the matrix is the average fluorescence from peduncle ROI’; ‘ROI_xLocAllFrames’ matrix (# of rows = # of neurons, # of columns = # of frames) contains the x position of the center of circular ROIs for individual neurons; ‘ROI_yLocAllFrames’ matrix (# of rows = # of neurons, # of columns = # of frames) contains the y position of the center of circular ROIs for individual neurons.
- https://cdn.elifesciences.org/articles/64108/elife-64108-fig4-data1-v1.zip
Figure 4—figure supplement 1
Single-cell correlation analysis.
(a) Fluorescence image of transgenic Hydra (#2) expressing GCaMP6s pan-neuronally. Individually tracked neuronal regions of interest (ROIs) are indicated with circles. The neurons are numbered in …
Figure 4—figure supplement 2
Single-cell correlation analysis.
(a) Fluorescence image of transgenic Hydra (#3) expressing GCaMP6s pan-neuronally. Individually tracked neuronal regions of interest (ROIs) are indicated with circles. The neurons are numbered in …
Figure 4—figure supplement 3
Random shuffling of single-cell correlation analysis.
Individual neuronal fluorescence time series were randomly shuffled to show the correlation coefficients were not due to random chance in all three Hydra (each row is a different Hydra). Original …
Videos
Video 1
Microfluidic system to study mechanosensory response in Hydra.
Dashed blue circle indicates the microfluidic valve that presses down on Hydra. Mechanosensory response from neurons and epitheliomuscular cells is shown.
Video 2
Spontaneous neural calcium activity in normal animals.
Dashed blue circle indicates the region of interest (ROI) used for calcium trace shown in blue (bottom) (playback 100×).
Video 3
Stimulated neural calcium activity in normal animals.
Dashed blue square indicates the region of interest (ROI) used for calcium trace shown in blue (bottom). Dashed white circle indicates the location of the valve that presses down on Hydra when …
Video 4
Spontaneous endodermal epitheliomuscular calcium activity in normal animals.
Dashed blue square indicates the region of interest (ROI) (entire frame) used for the calcium trace shown in blue (bottom) (playback 100×).
Video 5
Stimulated endodermal epitheliomuscular calcium activity in normal animals.
Dashed blue square indicates the region of interest (ROI) (entire frame) used for calcium trace shown in blue (bottom). Dashed white circle indicates the location of the valve that presses down on Hy…
Video 6
Spontaneous neural calcium activity in longitudinally bisected animals.
Dashed blue circle indicates the region of interest (ROI) used for calcium trace shown in blue (bottom) (playback 100×).
Video 7
Stimulated neural calcium activity in longitudinally bisected animals.
Dashed blue circle indicates the region of interest (ROI) used for calcium trace shown in blue (bottom). Dashed white circle indicates the location of the valve that presses down on Hydra when …
Video 8
Spontaneous neural calcium activity in headless animals.
Dashed blue circle indicates the region of interest (ROI) used for calcium trace shown in blue (bottom). Animal is less active without the head and fewer spikes in calcium activity of peduncle …
Video 9
Stimulated neural calcium activity in headless animals.
Dashed blue circle indicates the region of interest (ROI) used for calcium trace shown in blue (bottom). Dashed white circle indicates the location of the valve that presses down on Hydra when …
Video 10
Spontaneous endodermal epitheliomuscular calcium activity in footless animals.
Dashed blue square indicates the region of interest (ROI) (entire frame) used for the calcium trace shown in blue (bottom) (playback 100×).
Video 11
Stimulated endodermal epitheliomuscular calcium activity in footless animals.
Dashed blue square indicates the region of interest (ROI) (entire frame) used for calcium trace shown in blue (bottom). Dashed white circle indicates the location of the valve that presses down on Hy…
Video 12
Spontaneous endodermal epitheliomuscular calcium activity in headless animals.
Dashed blue square indicates the region of interest (ROI) (entire frame) used for the calcium trace shown in blue (bottom). Animal is less active without the head and fewer spikes in calcium …
Video 13
Stimulated endodermal epitheliomuscular calcium activity in headless animals.
Dashed blue square indicates the region of interest (ROI) (entire frame) used for calcium trace shown in blue (bottom). Dashed white circle indicates the location of the valve that presses down on Hy…
Video 14
Spontaneous endodermal epitheliomuscular calcium activity in bisected animals.
Dashed blue square indicates the region of interest (ROI) (entire frame) used for the calcium trace shown in blue (bottom). Animal is less active without the head and fewer spikes in calcium …
Video 15
Stimulated endodermal epitheliomuscular calcium activity in bisected animals.
Dashed blue square indicates the region of interest (ROI) (entire frame) used for calcium trace shown in blue (bottom). Dashed white circle indicates the location of the valve that presses down on Hy…
Video 16
Spontaneous endodermal epitheliomuscular calcium activity in body column animals.
Dashed blue square indicates the region of interest (ROI) (entire frame) used for the calcium trace shown in blue (bottom). Animal is less active without the head and fewer spikes in calcium …
Video 17
Stimulated endodermal epitheliomuscular calcium activity in body column animals.
Dashed blue square indicates the region of interest (ROI) (entire frame) used for calcium trace shown in blue (bottom). Dashed white circle indicates the location of the valve that presses down on Hy…
Video 18
Stimulated neural calcium activity in the hypostome and body column of normal animals.
Dashed blue circle indicates the region of interest (ROI) used for calcium trace shown in blue (bottom). Dashed white circle indicates the location of the valve that presses down on Hydra when …
Tables
Table 1
qPCR primers for neuronal subtypes.
| Subtype | transcriptID | F sequence | R sequence |
|---|---|---|---|
| en1 | t29955aep | GCC GCA GTA TCA TCA TAC AAA TC | CCA TAA ACC ACA CAT CGC ATA AA |
| en2 | t20666aep | CTT CTT GCT TCT ATC CTC GTT CT | TAC CTC AAG TAA ATT ATC GGT CTC G |
| en3 | t33579aep | CGT TGG TAT GAC TAT AAT CGT TGT TAT G | AGG ATA CAT CAC CCA CCA AAT C |
| ec1A | t22316aep | GCC TTT CTT TAT CTC GGG TAT CT | ACC TCC CAT GAG TAG CTG TA |
| ec1B | t20807aep | AAG ATC TAC GAC GTC ATA TCA ATC A | TCA TGC CCT TAT TAC CCT CTT G |
| ec2 | t7411aep | GCG CCT TGT AAC TAT GGT CTT A | TTC GTA GAA CAT TGT CAT CTT CCT |
| ec3A | t9620aep | CGG TGC TGC TCC TAA TTC AA | CTC CGG TGC ACT GAT TTA TAG G |
| ec3B | t7664aep | TGT TTC AAA TGC AGA CGA AGA TG | GCG TGT TTA TTT GCC TGG AC |
| ec3C | t19558aep | CGG TTA GAT ACA CTG CGG TTA G | TAC GTG CCG TTC TTC GTT T |
| ec4 | t33899aep | GGC TTT AAT CGT TGT AGC TCT TG | CTT GCT ATC TTC TGA CAA GTG ATT G |
| ec5 | t6329aep | CCA ACA ATG GTC GAA TGA AGA AA | ATC GCC AGG TTT GTA TCC TTT A |
| RP49 | t6797aep | GCC AAA CTG GAG AAA ACC TAA AG | TCA GGC ATA AGA TGA CGT GTC |
| Actin | t11116aep | CGC CCT CGT AGT TGA TAA TGG | AAT CCT TCT GTC CCA TAC CAA C |
