PDF-1 neuropeptide signaling modulates a neural circuit for mate-searching behavior in C. elegans

Abstract

Appetitive behaviors require complex decision making that involves the integration of environmental stimuli and physiological needs. C. elegans mate searching is a male-specific exploratory behavior regulated by two competing needs: food and reproductive appetite. We found that the pigment dispersing factor receptor (PDFR-1) modulates the circuit that encodes the male reproductive drive that promotes male exploration following mate deprivation. PDFR-1 and its ligand, PDF-1, stimulated mate searching in the male, but not in the hermaphrodite. pdf-1 was required in the gender-shared interneuron AIM, and the receptor acted in internal and external environment-sensing neurons of the shared nervous system (URY, PQR and PHA) to produce mate-searching behavior. Thus, the pdf-1 and pdfr-1 pathway functions in non-sex-specific neurons to produce a male-specific, goal-oriented exploratory behavior. Our results indicate that secretin neuropeptidergic signaling is involved in regulating motivational internal states.

Figure 1. PDF-1 neuropeptide signaling stimulates mate searching

(a) Genomic and protein domain structures of pdfr-1. (Top) Exons (black boxes) and introns (lines) of the pdfr-1 locus; (bottom) extracellular (red), 7 trans-membrane domains (black) and intracellular (blue) regions are shown. The changes in codon and amino acid produced by the bx142 mutation are indicated. (b) Graph shows P_L (probability of leaving food) values of wild type (wt), pdfr-1 (bx142) and (tm4457) mutants and mutants in the PDFR-1 ligands pdf-1(tm1996) and pdf-2(tm4393). The pdfr-1(tm4457) allele fails to complement bx142 indicating that the two mutations affect the same locus. The fosmid wrm0629dH07 contains the pdfr-1 locus. Error bars indicate SEM. n indicates total number of animals tested; (exp) indicates the number of independent population-based experiments. Maximum likelihood statistical analysis was used to compare P_L values, ***p<0.001, ns: no statistically significant difference (p≥0.05). (c) Graphs show the frequency of reversals and high angle turns (Ω turns) performed by wt and pdfr-1(bx142) mutant males during 5 minutes. Locomotion was observed on food and at two different time points off food: 1 minute after removal from food and 30 minutes after removal from food. n indicates total number of animals tested. Error bars indicate SEM. Statistical analysis was performed using Mann-Whitney test, ***p<0.001. (d) Graphs show the rate at which wild-type (wt) and pdfr-1(bx142) males travel 3.5 cm, from the starting point at the center of the plate within a food patch to the scoring distance 1 cm away from the edge of the assay plate. This was calculated as in the leaving assay, as P_L values (probability of reaching the scoring distance). Error bars indicate SEM. n indicates total number of animals tested; (exp) indicates the number of independent population-based experiments. Maximum likelihood statistical analysis was used to compare P_L values (probability of reaching the scoring distance), ***p<0.001, ns: no statistically significant difference (p≥0.05). (e) Graph shows P_L values of wild-type and pdfr-1(bx142) mutant males and wild-type hermaphrodites with or without the pdf-1 genomic overexpression (pdf-1 OE) transgene. npr-1(n1353) mutant males display rates of mate-searching behavior similar to wild-type males. Error bars indicate SEM; n indicates total number of animals tested; (exp) indicates the number of independent population-based experiments. Maximum likelihood statistical analysis was used to compare P_L values, ***p<0.001, ns: no statistically significant difference (p≥0.05).

Figure 2. The PDF-1/PDFR-1 pathway stimulates dispersal upon mate deprivation

(a) P_L values are shown for mutants in the insulin pathway: daf-16 encodes a forkhead transcription factor inhibited by insulin signaling and daf-2 encodes an insulin receptor. Error bars indicate SEM. n indicates total number of animals tested; (exp) indicates the number of independent population-based experiments. Maximum likelihood statistical analysis was used to compare P_L values, ***p<0.001, ns: no statistically significant difference (p≥0.05). (b) Percentage of quiescent worms in nutritionally high food (E. coli HB101). n indicates number of animals tested. χ² test was used for statistical analysis, ***p<0.001, **p<0.01, *p<0.05. (c) Frequency of reversals (during 5 minutes) was measured 1 minute and 30 minutes after removal from food. (d) Frequency of reversals (during 5 minutes) was measured off food 1 minute and 1 hour after removal from mates. Note that values in the first column of (c) and the second column of (d) are equivalent. Variability exists due to experiments being performed in different days for (c) and (d). In (c) and (d), n indicates number of animals tested, error bars indicate SEM and Mann-Whitney test was used for statistical analysis, ***p<0.001, **p<0.01, ns: no statistically significant difference (p≥0.05). (e) P_L values are shown for mutants in the food-sensing pathway. osm-9 encodes a TRPV channel expressed in amphid chemosensory neurons; eat-4 encodes a glutamate vesicular transporter expressed in amphid neurons; glr-1 encodes an AMPA glutamate receptor expressed in amphid interneurons. Error bars indicate SEM; n indicates total number of animals tested; (exp) indicates the number of independent population-based experiments. Maximum likelihood statistical analysis was used to compare P_L values, ***p<0.001.

Figure 3. Mate sensation and ray activity modulate the effects of PDF-1 signaling

The effects on mate-searching behavior (P_L values) of pdf-1 overexpression (pdf-1 OE) in wild-type males with and without mates and in mutants with defects in B-type ray neuron function are shown. lov-1 and pkd-2 encode TRP polycystins required for ray neuron function. Error bars indicate SEM; n indicates total number of animals tested; (exp) indicates the number of independent population-based experiments. Maximum likelihood statistical analysis was used to compare P_L values. ***p<0.001, **p<0.01, ns: no statistically significant difference (p≥0.05).

Figure 4. pdfr-1 is required in sensory neurons URY, PQR and PHA to produce mate-searching behavior

(a–e) Expression of P(pdfr-1)::RFP in males at the L4 stage. (a and d) DIC images, (b, c and e) fluorescence images. Neurons in the head (a, b) and the retro-vesicular ganglion (c) labeled with a pdfr-1::NLS RFP reporter. (d and e), neurons in the tail labeled with a pdfr-1::RFP reporter. Anterior is to the left in all images. (a, b, d and e) lateral views; (c) ventral view. Scale bars, 5 μm (f) Rescue of mate-searching behavior in pdfr-1(bx142) mutants with a heat-shock inducible promoter driving isoforms b and d. Lines indicate independent arrays of the transgene. Graphs show the proportion of males that left food after 3 hours of leaving assay. n indicates number of animals tested; χ² test was used for statistical analysis, ***p<0.001, ns: no statistically significant difference (p≥0.05). (g) Effects of laser ablation of URY, PQR and PHA on mate-searching behavior in wild type and pdfr-1(bx142) males. The rate of leaving in non-ablated control wild-type males was slightly reduced by the presence of the transgenes used to identify the target neurons. Error bars indicate SEM; n indicates total number of animals tested; (exp) indicates the number of independent population-based experiments. Maximum likelihood statistical analysis was used to compare P_L values. **p<0.01, ns: no statistically significant difference (p≥0.05).

Figure 5. The interneuron AIM is a source of PDF-1 for mate-searching behavior

(a–d) Expression of P(pdf-1)::RFP in adult males. (a and c) DIC images, (b and d) fluorescence images of neurons in the head (b) and the tail and posterior ventral cord (d). All images show a ventral view, anterior to the left. Scale bars, 5 μm. (e) Effects on mate-searching behavior (P_L values) of ablation of all pdf-1-expressing male-specific neurons, AVB or AIM in wild-type males. Rescue of mate-searching behavior in pdf-1(tm1996) mutants by expression of a pdf-1 cDNA in AIM. Error bars indicate SEM; n indicates total number of animals tested; (exp) indicates the number of independent population-based experiments. Maximum likelihood statistical analysis was used to compare P_L values, ***p<0.001, **p<0.01, ns: no statistically significant difference (p≥0.05).