C. elegans dopaminergic D2-like receptors delimit recurrent cholinergic-mediated motor programs during a goal-oriented behavior

Abstract

Caenorhabditis elegans male copulation requires coordinated temporal-spatial execution of different motor outputs. During mating, a cloacal circuit consisting of cholinergic sensory-motor neurons and sex muscles maintains the male's position and executes copulatory spicule thrusts at his mate's vulva. However, distinct signaling mechanisms that delimit these behaviors to their proper context are unclear. We found that dopamine (DA) signaling directs copulatory spicule insertion attempts to the hermaphrodite vulva by dampening spurious stimulus-independent sex muscle contractions. From pharmacology and genetic analyses, DA antagonizes stimulatory ACh signaling via the D2-like receptors, DOP-2 and DOP-3, and Gα(o/i) proteins, GOA-1 and GPA-7. Calcium imaging and optogenetics suggest that heightened DA-expressing ray neuron activities coincide with the cholinergic cloacal ganglia function during spicule insertion attempts. D2-like receptor signaling also attenuates the excitability of additional mating circuits to reduce the duration of mating attempts with unproductive and/or inappropriate partners. This suggests that, during wild-type mating, simultaneous DA-ACh signaling modulates the activity threshold of repetitive motor programs, thus confining the behavior to the proper situational context.

Figure 1. DA is required for spicule insertion during mating.

The number of males tested and the percentage of potent males is listed at the bottom and top of the bars, respectively. (A) Mating potency. (B–D) Males were mated with paralyzed mates. (B) Percent of males displaying spicule related behaviors. (C) The duration in contact with a mate between insertion attempts. (D) The spicule efficiency index. Symbols represent an individual male performance. Open symbols represent unsuccessful insertions. Line and error bars represent mean and SEM.

Figure 2. DA inhibits ACh-agonist-induced spicule protraction.

The number of males tested and the percentage of spicule protracted males is listed at the bottom and top of the bars, respectively. (A&B) One-day-old virgin males were exposed to 30 mM DA and either LEV, NIC, OXOM or ARE. (A) DA simultaneous exposure with or (B) followed by (fb) ACh agonists. (C) Males were bathed in each drug combination for increasing duration. p-values determined with Fisher's exact test comparing mutants to wild type. MIX = LEV,NIC, OXO.

Figure 3. Ca⁺² transients in DA ray neurons increase during arched postures.

% ΔF/F0 trace for 5 seconds. Representative recordings for individual wild-type males with (A) an arched posture during spicule insertion attempts (left), (B) with non-arched posture during spicule insertion attempts (left), and (C) arched scanning posture (left). Non-arched scanning recordings for each male are displayed on the right (A–C). A Pdat-1:unc-103(gf) transgenic male displaying an arched posture at the vulva (left) and during scanning (right) (D). The In-Contact Length % (ICL%) are the numbers located at the top of each bar taken from a representative frame for each 1 sec intervals.

Figure 4. Activation of cloacal neurons increase Ca⁺² transients in Rn7A, and activation of DA neurons attenuate ARE-induced muscle contraction via D2-like receptors.

(A) Abridged schema of synapses between Rn5A, 7A, 9A and spicule circuit components. Arrows embedded in bars indicate reciprocal electrical and chemical synapses. Connections relevant to this work are depicted. For the complete wiring, refer to the male wiring project (S.W. Emmons, D.H. Hall, M. Xu, Y. Wang and T. Jerrel, Male Wiring Project, Albert Einstein College of Medicine, http://worms.aecom.yu.edu/pages/male_wiring_project.htm, [50]). Gubernacular erector (GER), gubernacular retractor (GRT), anterior oblique (AOB), posterior oblique (POB), dorsal spicule protractor (DSP), ventral spicule protractor (VSP) and anal depressor (ADP). (B) Video montage depicting changes in G-CaMP fluorescence in Rn7A. (C) The average %ΔF/F0 determined from all tested males used in both samples (top) with (n = 14) or (bottom) without (n = 11) ATR treatment representing Rn7A neuron Ca⁺² transients before, during and after PCB, SPC stimulation. The dark and light blue lines represent the average and standard deviation values, respectively. (D) Males that protracted their spicules during simultaneous blue light stimulation of DA neurons and ARE exposure. The genotypes are written below each bar. The number of males tested and the percentage of spicule protracted males are listed at the bottom and top of the bars, respectively.

Figure 5. D2-like signaling promotes spicule insertion attempts.

(A) Duration of vulval contact until spicule insertion or 120 sec. Symbols represent individual male performance. Open symbols represent unsuccessful insertions. (B) Duration over the vulval slit during the 1^st spicule insertion attempt. For A&B, line and error bars represent mean and SEM. (C) Spicule movement frequency calculated for 6 sec during spicule thrust against vulval slit. Symbols represent an individual frequency interval. Line and error bars represent mean and SD.

Figure 6. D2-like signaling promotes mating fitness.

(A) Y-axis depicts the number of pregnant females amongst 1, 5 or 20 potential mates. Symbols represent individual male's sexual prowess. (B) Mating competition test pairing a wild type and a mutant male with one fog-2 female +/−10 paralyzed males (first 4 columns), and number of fog-2 females impregnated when wild type and dop-2; dop-3 males were paired individually with 10 C. briggsae hermaphrodites ( last 2 columns). The top and bottom of each column indicates the % of pregnant females and the number of males that copulated or number of males assayed (last 2 columns). (C) The cumulative time in contact with males that a wild type and dop-2; dop-3 male, represented by each symbol, spent when surrounded by 40–50 paralyzed males. Each data subset represents non-ablated animals (mock), PCB or PCS ablated animals. p-values calculated using the Mann-Whitney test. For A&C, line and error bars represent mean and SEM. (D) Top shows coincident D2-like and ACh signaling during spicule insertion attempts. Bottom depicts residual DA signaling when a male moves off the vulva. Blue and green color represents DA and ACh signaling components, respectively.