A cholinergic-regulated circuit coordinates the maintenance and bi-stable states of a sensory-motor behavior during Caenorhabditis elegans male copulation

Abstract

Penetration of a male copulatory organ into a suitable mate is a conserved and necessary behavioral step for most terrestrial matings; however, the detailed molecular and cellular mechanisms for this distinct social interaction have not been elucidated in any animal. During mating, the Caenorhabditis elegans male cloaca is maintained over the hermaphrodite's vulva as he attempts to insert his copulatory spicules. Rhythmic spicule thrusts cease when insertion is sensed. Circuit components consisting of sensory/motor neurons and sex muscles for these steps have been previously identified, but it was unclear how their outputs are integrated to generate a coordinated behavior pattern. Here, we show that cholinergic signaling between the cloacal sensory/motor neurons and the posterior sex muscles sustains genital contact between the sexes. Simultaneously, via gap junctions, signaling from these muscles is transmitted to the spicule muscles, thus coupling repeated spicule thrusts with vulval contact. To transit from rhythmic to sustained muscle contraction during penetration, the SPC sensory-motor neurons integrate the signal of spicule's position in the vulva with inputs from the hook and cloacal sensilla. The UNC-103 K(+) channel maintains a high excitability threshold in the circuit, so that sustained spicule muscle contraction is not stimulated by fewer inputs. We demonstrate that coordination of sensory inputs and motor outputs used to initiate, maintain, self-monitor, and complete an innate behavior is accomplished via the coupling of a few circuit components.

Figure 1. Ionotropic ACh receptors are required for sustained vulval contact.

(A) Relative mating potency of mutant males normalized to wild type. The number of males assayed for each strain is listed within each bar. The number above the bar refers to the normalized mating potency value. The males were mated with free-moving hermaphrodites. Each mutant strain was compared to the wild-type males that were assayed in parallel, using the Fisher's exact test. Asterisks (***) indicate the p value <0.0001. (B) Profiles of the vulval contact duration. The males were mated into paralyzed hermaphrodites. The average duration of vulval contact for each male was normalized to the wild type mean to obtain the “normalized score for vulval contact duration”, as a measurement of the male's ability to maintain precise vulval contact (Materials and Methods). The mean of the normalized score for vulval contact duration for each strain is represented by the height of the bar, and it is also indicated by the number below the top of the bar. Error bars indicate the standard error of the mean. The number of males assayed for each strain is listed within each bar. Each mutant strain was compared to wild-type males using the Mann-Whitney non-parametric test. Asterisk (*) indicates the p value <0.005. The raw values of average duration of vulval contact for each male tested are presented in Figure S2A.

Figure 2. Male tail expression of acr-16 and unc-29.

Gubernacular erector (GER), gubernacular retractor (GRT), anterior oblique (AOB), dorsal spicule protractor (DSP), ventral spicule protractor (VSP) and anal depressor (ADP) muscle. (A–D) Fluorescence images of the tail region of adult males expressing the Pacr-16:YFP construct. Scale bar, 20 µm. (E–H) Fluorescence images of the tail region of adult males expressing the Punc-29:unc-29::YFP construct. Scale bar, 20 µm.

Figure 3. Circuit diagram of cells that control spicule movement and model of prolonged vulval contact coupling to rhythmic protractor contractions.

Gubernacular erector (GER), gubernacular retractor (GRT), anterior oblique (AOB), posterior oblique (POB), dorsal spicule protractor (DSP), ventral spicule protractor (VSP) and anal depressor (ADP). Arrows and bars indicate chemical synapses and gap junctions, respectively. Bi-directional arrows refer to cells that make reciprocal chemical synapses. Bi-directional arrows embedded in bars refer to cells that make gap junctions in addition to reciprocal chemical synapses. The green circles refer to cholinergic neurons. (A) Bilateral electrical and chemical connectivity diagram of male neurons and muscles that control the movements of the spicules [adapted from , and the Male Wiring Project]. (B) Abbreviated cartoon of limited connections between neurons and muscles discussed in this work. Refer to (Male Wiring Project, Albert Einstein College of Medicine, http://worms.aecom.yu.edu/pages/male_wiring_project.htm) for a more complete list of connections to other cells in the male. (C) Cartoon depicting changes in male tail muscles upon vulval contact. Left panel depicts relative locations and electrical junctions between the male sex muscles. The right panel depicts the shortening of the AOB and POB muscles causing a curvature in the posterior male tail (red arrow) upon chemical stimulation from the PCA, PCB and PCC neurons. Stimulated GER muscles relay their signals to the spicule protractor muscles causing rhythmic contractions (blue arrow).

Figure 4. The postcloacal sensilla (p.c.s.) neurons and the oblique muscles facilitate prolonged vulval contact.

(A–C) The average duration of vulval contact for each male was normalized to the mock-ablated mean to obtain the “normalized score for vulval contact duration” (Materials and Methods). The mean of normalized score for each strain is represented by the height of the bar, and it is also indicated by the number below the top of the bar. Error bars indicate the standard error of the mean. The number of males assayed for each operation is listed within each bar. Ablated males are compared to mock-ablated males that mated with the same group of hermaphrodites. Data that are shown in the same panel were obtained in parallel, but cannot be compared directly to data presented in different panels. (D) Ablation of the gubernacular muscles increases the duration of sperm release. The average duration of sperm release is represented by the height of the bar, and it is also indicated by the number below the top of the bar. The number of males assayed for each operation is listed within each bar. Error bars indicate the standard error of the mean. (E) Restoring unc-29 in male muscles reverses the vulval contact defect of the locomotion-restored unc-29(lf);acr-16(0) males. The average duration of vulval contact for each male was normalized to the mean of the body wall muscle unc-29 rescued males, to obtain the “normalized score for vulval contact duration”. The mean of normalized score for each strain is represented by the height of the bar, and it is also indicated by the number below the top of the bar. The number of males assayed for each operation is listed within each bar. Error bars indicate the standard error of the mean. Asterisks (**) indicate the p value <0.005, (*) indicates the p value <0.05, calculated using the Mann-Whitney non-parametric test. The raw values of average duration of vulval contact or duration of sperm release for each male tested are presented in Figure S2B-S2F.

Figure 5. Stimulation of the gubernacular-oblique muscle group induces repetitive spicule thrusts and Ca²⁺ transients in the spicule protractor-anal depressor muscles.

(A) Displacement of the spicule during and between brief blue light pulses. The grey regions indicate the time periods of blue light pulses. An image sequence of the male tail was captured for ∼30 seconds when light pulses were applied repetitively. A region of interest (ROI) was placed at the base of a spicule, and the standard deviation of the pixel intensity within the ROI was obtained to indicate spicule displacement (see Materials and Methods). A representative trace for males that expressed active ChR2 (in the presence of all-trans retinal) is shown in the upper panel; a representative trace for males that expressed inactive ChR2 (in the absence of all-trans retinal) is shown in the lower panel. a.u. arbitrary units. (B) Raw fluorescence and corrected intensity traces for a representative male. Upper panel: raw fluorescence intensity traces in the protractor without gubernacular-oblique muscle stimulation for the baseline recording (with inactive ChR2), and during stimulation of active ChR2 on the gubernacular-oblique muscle group. Fluorescence intensity for both G-CaMP and DsRed channels are plotted, as well as the background fluorescence intensity for each channel. The periods that the light stimuli were applied are indicated by grey bars; the numbers on the X-axis indicate the time points since the onset of the most recent light stimulus; the 30 minute incubation is indicated on the X-axis. a.u. arbitrary units. Lower panel: corrected G-CaMP fold change trace during light stimulation with active ChR2. (C) Maximal G-CaMP intensity changes in the protractors during light stimulation. The number of males assayed for each operation is listed at the bottom of each bar. The numbers below the top of the bars indicate the mean of maximal G-CaMP intensity changes of the population. Error bars indicate the standard error of the mean. Asterisk (***) indicate the p value <0.0005, (*) indicates the p value <0.05, calculated using the Mann-Whitney non-parametric test. (D) Average G-CaMP intensity changes before, during and after a pulse of blue light stimulation. The blue arrow indicates the beginning of light stimulation, the red arrow indicates the onset of G-CaMP intensity increase, and the black arrow indicates the end of light stimulation. The trace in red represents males with active ChR2. Males without active ChR2 are represented by the blue trace. The grey region around each curve represents the standard error of the mean. 4 males were measured for each trace. (E) Averaged G-CaMP intensity change traces in the anal depressor muscle (ADP), the gubernacular erector (GER) and a posterior body wall muscle (BW), upon laser microbeam stimulation of the gubernacular erector muscle or the posterior body wall muscle in the male tail. Traces represent averaged G-CaMP intensity changes in specific muscles. The grey region around each trace represents the standard error of the mean. Left panel, the gubernacular erector on one side of the males was stimulated by a laser microbeam. The energy of the laser was adjusted to the lowest level that could elicit Ca²⁺ transients in the muscles. N = 10 males. Right panel, a body wall muscle that has no gap junction to the protractor-anal depressor muscle group was stimulated by a laser microbeam. Ca²⁺ transients in the gubernacular erector and the anal depressor muscles were detected by using G-CaMP. Stimulation of the posterior body wall muscle failed to induce Ca²⁺ transients in the anal depressor muscle, indicating that in the left panel, Ca²⁺ transients in the anal depressor muscle were not caused by non-specific laser damage in the male tail. N = 11 males. (F) Effects of innexin mutant, RNAi of innexins and SPC ablation on ChR2-induced spicule thrusts. The unc-9(lf) males were compared to the wild type. The innexin RNAi strains with a wild-type genetic background were compared to the wild type that was fed with empty vector. The innexin RNAi strains with an unc-9(lf) genetic background were compared to the unc-9(lf) that was fed with empty vector-containing bacteria. The SPC-ablated wild type was compared to un-operated wild type. The numbers above the bars show the percentage of males that displayed rapid spicule thrusts upon light stimulation. The number of males assayed for each strain is listed within the bar. Asterisks (***) indicate the p value <0.0001, using the Fisher's exact test.

Figure 6. Stimulation of ChR2 in different spicule circuit components.

(A) Left panel, a male that expresses Pgar-3:ChR2::YFP holds his spicules inside of his tail prior to illumination. Right panel, the blue light induces sustained spicule protraction of this male. (B) Sustained spicule protraction induced by stimulation of ChR2 in different circuit components. The numbers on the vertical axis represent the percentage of males that protracted their spicules when different circuit components were activated by light-stimulated ChR2. The numbers above the bars show the percentage of males that protracted their spicules. The number of males assayed for each strain is listed within the bar. Asterisks (***) indicate the p value <0.0001, (**) indicate the p value <0.001, using the Fisher's exact test.