Caenorhabditis elegans male sensory-motor neurons and dopaminergic support cells couple ejaculation and post-ejaculatory behaviors
- Howard Hughes Medical Institute, Texas A&M University, United States
Figures
Figure 1
Conceptual diagram of structures and connectivity in the C. elegans male tail.
ger = gubernaculum erector muscle, grt = gubernaculum retractor muscle, adp = anal depressor muscle, ob = oblique muscle. (A) Diagram of the male tail positioned at the hermaphrodite vulva. The …
Figure 2
Conceptual diagrams of the structure and connectivity involved in ejaculation.
(A) Diagram of the males’ reproductive tract. dpm = dorsal protractor muscle, vpm = ventral protractor muscle. (B) Diagram of the initiation step of ejaculation. When the valve region separating the …
Figure 3
The refractory period is regulated by sperm release.
Line represents median. (A) 1st insert: the time required for a male to insert his copulatory spicules into the hermaphrodite vulva from the time he was placed with the hermaphrodites. 2nd insert: …
Figure 4 with 2 supplements
The spicule sensilla promote insertion behaviors.
(A) Sperm transfer ranking for males lacking the SPV and SPD sensory neurons and their age-matched controls. A male received a 0 if he ejaculated into the uterus, a 1 if he inserted but didn't …
Figure 4—figure supplement 1
Cells affected by cutting off the spicule tips with a laser.
(A) Simplified diagram of the neuronal connectivity present in the spicule circuit in the male tail. (B) The same diagram as (A) representing what cells (with an ‘X’ over them) and connections are …
Figure 4—figure supplement 2
Spicule tips cut males display defects in mating.
(A) Mating drive in spicule tip cut males. The y-axis is the time it takes a virgin male to commence backing along a hermaphrodite cuticle. (B) Total time at vulva prior to insert in operated …
Figure 5 with 2 supplements
Ca2+ transients in SPV, SPD, SPC neurons, and gonad muscles during intromission and ejaculation.
(A, B, D). % ΔF/F0 trace including insertion and ejaculation. One representative recording is reported for each cell type. x-axis is time (seconds), y-axis is % ΔF/F0. The time of mating behaviors …
Figure 5—figure supplement 1
Ca2+ transient changes in cells that regulate ejaculation.
% ΔF/F0 trace for mating from insertion through sperm release and retraction, except for the first trace in each column which represents vulva prodding behavior. Arrows indicate when the behavior …
Figure 5—figure supplement 2
Removing the spicule sensilla impacts the Ca2+ changes in the gonadal valve.
% ΔF/F0 trace for mating from insertion (1) through valve opening (2) and sperm release (3). G-CaMP is expressed in the valve via Ptry-5. (A) Intact males and (B) spicule tip ablated males.
Figure 6 with 1 supplement
Cholinergic and glutamatergic neurons promote ejaculation.
(A) Chart of promoter, expression pattern, and ability of the promoter driving ChR2 to induce ectopic ejaculation (Ejac). (B) Graph of ectopic ejaculation in response to 475 nm wavelength light in …
Figure 6—figure supplement 1
% of ChR2-expressing males that ejaculate in response to 475 wavelength light stimulation.
(A) Rate of ectopic ejaculation in response to 475 nm wavelength light exposure in males expressing ChR2 in the spicule and post cloacal sensilla. x-axis lists the cells ablated, y-axis is the % of …
Figure 7 with 1 supplement
PCA contributes to the sex muscle-controlled spicule movement required for sperm to drain from the cloaca into the uterus.
(A) Images of sex muscle activity during intromission and ejaculation. Sc = spicule, ger = gubernaculum erector, adp = anal depressor, grt = gubernaculum retractor. The gubernaculum is located …
Figure 7—figure supplement 1
Ca2+ transient changes in sex muscles and the PCA neurons during ejaculation.
(A) Ca2+ transient changes in the muscles during mating. % ΔF/F0 trace for mating from insertion (1) through sperm release (3), and retraction (4). (A) G-CaMP is expressed in the sex muscles via …
Figure 8 with 4 supplements
Calcium imaging in spicule tip cut males.
(A) % ΔF/F0 in the PCA. (B) % ΔF/F0 in the dorsal protractor, anal depressor, and gubernaculum erector and retractor. (C and D) % ΔF/F0 in the SPC. (E) % ΔF/F0 in the PCB.
Figure 8—figure supplement 1
Removing the spicule sensilla has no effect on the PCA neuron Ca2+ transients.
% ΔF/F0 for 30 s following insertion in PCA neurons. G-CaMP expressed in PCA using Peat-4. Males were either operated on by having their spicule tips removed (Spicule tips cut column) or exposed to …
Figure 8—figure supplement 2
Removing the spicule sensilla has no effect on the sex muscle Ca2+ transients.
Calcium imaging in the dorsal sex muscles (dorsal protractor, gubernaculum erector and retractor, anal depressor) in spicule tip cut (right column) and non-cut controls (left column). G-CaMP …
Figure 8—figure supplement 3
Ca2+ transients increase at a slower rate in the SPC neurons when the spicule sensilla are removed.
Calcium imaging in the SPC in spicule tips cut males (right column) and non-operated control males (left column). G-CaMP expressed from Pgar-3B. % ΔF/F0 reported from spicule insertion into …
Figure 8—figure supplement 4
Removing the spicule sensilla has no effect on the PCB neuron Ca2+ transients.
Calcium imaging from Pdop-2:G-CaMP expressed in PCB. The first column shows the % ΔF/F0 for insertion and sperm release for five males. The final graph shows calcium imaging in the PCB while the …
Figure 9 with 2 supplements
Socket cell dopamine (DA) promotes sperm release.
(A) % ΔF/F0 in the socket cells. G-CaMP is expressed from Pcat-2. (B) % ΔF/F0 in the dopaminergic rays 5,7,9A. G-CaMP is expressed from Pdat-1. (C) % of males that transferred sperm for ≥10 s into …
Figure 9—figure supplement 1
Socket cell DA regulates male mating.
(A) Dopamine synthesis pathway. Two DA synthesis genes, bas-1 and cat-2, are expressed in the male socket cells. Left images are DIC, right fluorescence. Sp = spicule, So = socket cells. Dorsal is …
Figure 9—figure supplement 2
Ca2+ transients in the DA-expressing cells in the male tail.
(A and B) The first panel is the % ΔF/F0 in a male continuously prodding at the hermaphrodite vulva. All subsequent panels are % ΔF/F0 in males during insertion and sperm transfer into the uterus. (A…
Figure 10
Socket cell DA and neuronal ACh regulate ejaculation and the refractory period.
(A) The steps of C. elegans male mating behavior. The order of the individual steps is given by the numbers. The refractory period is a period of reduced activity and mating ability following …
Tables
Table 1
Ca2+ transients following spicule insertion
| Cell | ↑Ca2+ insertion→peak (sec)* | Significance | Slope of initial Ca2+ increase (ΔF/F0%/sec) | Significance |
|---|---|---|---|---|
| SPC | 1.3 ± 0.52a | 45 ± 13 | ||
| Valve | 1.8 ± 0.79a | 60 ± 14 | ||
| SPV/SPD | 4.0 ± 1.0 | p<0.05 to ‘a’ | 21 ± 14 | p<0.05 to SPC and valve |
| PCA | 6.3 ± 0.56 | p<0.05 to ‘a’ | 15 ± 2.9 | p<0.05 to SPC and valve |
| Socket cells | 1.7 ± 0.36a | 94 ± 18 | p<0.005 to PCA | |
| Sex muscles 1st peak | 1.3 ± 1.7a | 155 ± 82 | p<0.005 to socket cells | |
| Sex muscles 2nd peak | 11 ± 3.9 | p<0.05 to sex muscles 1st peak | 46 ± 19† | p<0.0001 to sex muscles 1st peak |
| SPC control | 1.2 ± 0.30 | 60 ± 26 | ||
| SPC ablated | 2.2 ± 0.74 | p=0.022 to SPC control | 29 ± 12 | p=0.035 to SPC control |
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Mean and standard deviation reported. For non-operated cell types, n = 5. Results of ANOVA: Newman–Keuls Multiple Comparison Test are shown. SPC–control and ablated, Mann–Whitney t test. SPC–control n = 6, SPC–ablated n = 7.
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*
Time (sec) required for Ca2+ to increase from spicule insertion to Ca2+ peak.
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†
Slope determined from where the Ca2+ begins to increase for a second time to Ca2+ peak following this second increase.
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a
Peak times that are significantly different from the SPV/SPD and PCA cells.
