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Behavioral devaluation by local resistance to dopamine

Nature Neuroscience volume 28, pages 2493–2501 (2025)Cite this article

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Abstract

Repeated experiences can cause behavior-specific fatigue. We use Drosophila to study this common form of motivational change, finding that prior matings make males more likely to abandon future copulations when challenged. Here we show that, during mating, dopamine signals through the D2-like receptor (D2R) to promote resilience to challenges that might otherwise cause the male to switch behaviors. This motivating dopamine signal suppresses the output of the copulation decision neurons (CDNs), which can truncate matings when pushed past threshold. Repetition-induced devaluation of mating results from β-arrestin-dependent desensitization of the D2R on the CDNs, rendering them temporarily resistant to naturally released or experimentally supplied dopamine. When local desensitization to dopamine is prevented, the male shows no signs of fatigue, treating each mating as if it were his first. These findings explain a widespread motivational phenomenon and reveal a natural function for the notorious susceptibility of the D2R to drug-induced desensitization.

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Fig. 1: Dopamine released during copulation decreases the resiliency of future matings.
Fig. 2: Dopamine protects mating by signaling through the D2R to the CDNs.
Fig. 3: Matings desensitize the CDNs to future dopamine.
Fig. 4: Gαo signaling promotes persistence during current matings but cannot induce the devaluation of future matings.
Fig. 5: The classical GPCR inactivation pathway is required to implement consummatory satiety.
Fig. 6: Behavioral devaluation by local resistance to dopamine.

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Data availability

Source data for all figures and extended data figures are available as Supplementary Information. Source data are provided with this paper.

Code availability

All codes used in this paper can be found at https://github.com/CrickmoreRoguljaLabs. See Supplementary Table 5 for additional information.

Jeffery’s confidence interval: https://github.com/CrickmoreRoguljaLabs/UpdatedJeffi

Copulation duration code: https://github.com/CrickmoreRoguljaLabs/FlyKnight

FLIMage software: https://github.com/ryoheiyasuda/FLIMage_public

MATLAB calcium imaging analysis code (V7): https://github.com/CrickmoreRoguljaLabs/GCaMPfromFLIMage

Python FLIM analysis code: https://github.com/CrickmoreRoguljaLabs/flim-analysis

MATLAB GCaMP normalized to tdTomato code: https://github.com/CrickmoreRoguljaLabs/GCaMP-tdt_Analysis

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Acknowledgements

We thank S. Thornquist and G. Maimon for UAS-eOPN3; the Rogulja and Crickmore laboratories for discussions and comments on the manuscript; E. Glantz for the design of the behavioral arenas for copulation duration with thermogenetics; K. Thieringer, L. Kerrick, R. Persaud, P. Rifkin and A. Antony for assistance with experiments; M. Pitsch for help analyzing single-cell sequencing data; O. Mazor and P. Gorelik (Harvard Medical School Neuroinstrumentation Core) for designing the thermosensation assay and for technical advice; the IDDRC Cellular Imaging Core, funded by National Institutes of Health (NIH) P50 HD105351; and T. Schwarz, J. Kaplan, B. Ferguson, S. Flavell, L. Goodrich, S. Zhang and K. Lezgiyeva for discussions. This work was funded by the NIH (R01NS111441 and R01GM134222) and the Harvard Brain Initiative. L.E.M. was supported by a National Science Foundation Graduate Research Fellowship (DGE 2140743).

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Authors and Affiliations

  1. F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

    Lauren E. Miner, Aditya K. Gautham & Michael A. Crickmore

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  1. Lauren E. Miner
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  2. Aditya K. Gautham
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  3. Michael A. Crickmore
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Contributions

L.E.M. performed the experiments, with early assistance from A.K.G. L.E.M. and M.A.C. wrote the manuscript, with input from A.K.G. All authors designed experiments and analyzed results.

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Correspondence to Michael A. Crickmore.

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Nature Neuroscience thanks Toshihide Hige and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Consummatory satiety is a function of time spent mating.

(A) The percentage of males engaged in mating behaviors – either courtship or copulation – declines as they spend more time mating ad libitum with females in the satiety assay (n = 36, 6 groups of 6). (B) The fraction of males terminating matings in response to the same heat threat increases with time spent in the satiety assay (n = 10-38). (C) Copulation duration does not change with satiety (n = 16-25). (D) A thermosensation assay gives a group of ~10 flies a choice between a heated area and a room temperature area in an open arena. Schematic created with the assistance of BioRender. Miner, L. (2025) https://BioRender.com/m76b723. (E) Male Drosophila will avoid the area of a thermosensation assay heated to 28°C or greater, whether or not they have recently mated (n = ~10/group, 3-6 groups per test temperature). (F) Effectiveness of the satiety assay is independent of the type of female used, so long as the females are receptive. Spending time with unreceptive females does not induce consummatory satiety (n = 23-29). (G) Thermogenetic stimulation of Corazonin neurons for 4.5 hours causes ejaculation and reduces the ejaculatory bulb volume (n = 24-25). (H) Representative images of ejaculatory bulb scores. The lumen that stores sperm and seminal fluid is pseudocolored pink. (I) Unlike stimulation of the dopamine neurons in Fig. 1d, 4.5 hours of thermogenetic pre-stimulation of the Corazonin neurons does not induce an increase in the fraction of mating pairs terminating in response to a heat threat (n = 24-25).(J) Silencing the male Corazonin neurons throughout a satiety assay prevents sperm transfer from occurring during mating, shown by a still full ejaculatory bulb after the assay (n = 20 each). Note that matings with the male Crz neurons silenced are extended to ~90 mins each. (K) Consummatory satiety is still observed when ejaculation was prevented during prior matings. Note that the silencing was turned off during the final, challenged mating (n = 27-30).

Source data

Extended Data Fig. 2 Testing courtship circuit elements for potential roles in consummatory satiety.

(A) Males are group-housed in standard food vials and exposed to either warmth to activate TrpA1 or left at room temperature for 4.5 hrs, then their motivation is assessed by testing the probability that they terminate the mating in response to a bright blue light stressor in a subsequent mating. See Methods for explanation of stimulation temperatures. (B) Model: known circuit elements controlling appetitive satiety and courtship behavior. (C) Pre-stimulation of known courtship circuitry neurons does not induce increased mating termination in response to a pulse of blue light (n = 7-41). The high baseline in VT02857 precluded us from testing this Gal4 line, which labels the sexually dimorphic brain dopamine neuron called aSP4 (ref. 5). (D) Pre-stimulation of the Copulation Reporting Neurons (CRNs; R42G02-Gal4), a putative sensory neuron population that projects to the genitalia and has been reported to detect mating16, induces increased termination in response to a pulse of blue light (n = 31-33). (E) Pre-stimulation of the CRNs does not alter copulation duration (n = 27 each).

Source data

Extended Data Fig. 3 Dopaminergic release during mating is transiently protective while inducing long-term deprioritization of future copulations.

(A) Parental controls for Fig. 1d, exposure to the heat treatment used to thermogenetically activate TrpA1 does not itself induce a devaluation of mating (n = 25-33). (B) Pre-stimulation of dopamine neurons does not change copulation duration (n = 14-17). (C) Sustained stimulation of dopamine neurons during mating extends copulation duration (n = 17-22). (D) Stimulation of dopamine neurons reduces the probability of the mating terminating in response to a heat threat (n = 15-38). (E) Electrical silencing of dopamine neurons during mating in a satiety assay does not alter the number of times males mate in the assay (n = 13-25). (F) Sustained silencing of dopamine neurons does not alter copulation duration (n = 18-23). (G) Silencing dopamine neurons after a satiety assay is not sufficient to counteract the effects of satiety on motivation (n = 8-10).

Source data

Extended Data Fig. 4 Dopamine signaling to the CDNs through the D2R supports mating persistence.

(A) Of the four dopamine receptors in Drosophila, whole-animal deletion of the D2 receptor most strongly increases termination in response to heat threats (n = 27-33). Note: we observed a courtship deficit and low mating rate in D2R mutant flies when conducting these experiments. We had not previously detected a role for this gene in courtship using a p-element insertion5,17. (B) Mutation of any of the four known dopamine receptors in Drosophila males has little effect on copulation duration (n = 10-15). (C) Electrical silencing of the dopamine neurons increases termination in response to a heat threat, an effect that is blocked by CDN silencing (n = 26-33). (D) Deletion and rescue of D2R expression in the CDNs does not alter copulation duration (n = 19-36). (E) Expression of UAS-D2R in the CDNs does not decrease the fraction of mating pairs terminating in response to a heat threat delivered at 10 minutes (n = 27-32). (F) RNAi-mediated knockdown of D2R in the CDNs does not alter copulation duration (n = 12-17). (G) Knockdown of the Dop1R1, which is expressed in some but not all CDNs, also increased termination in response to a heat threat at 8 mins into mating (n = 24-30).

Source data

Extended Data Fig. 5 Physiological evidence for D2R mediating the action of dopamine on the CDNs.

(A) Example ROIs of the CDN axons used for our intensity and fluorescence lifetime imaging experiments. The CDNs project bilaterally, we only use one ROI on one side per animal for our experiments. (B) In the absence of ChR2-XXM driven stimulation, the effects of dopamine on CDN calcium are less obvious (n = 10 each). (C) Example experiment where ChR2-XXM is used to elevate CDN activity before bath applying dopamine (left: average trace of raw photon counts displayed as mean ± SEM, middle: normalized individual traces, right: normalized average trace displayed as mean ± SEM). Because the initial rise in calcium is variable, we wait until calcium levels stabilize before perfusing and use the 10 s immediately preceding the start of perfusion as a baseline. Traces in the main figures have been cropped to only show activity once calcium levels have stabilized. Control animals without perfusion are shown (n = 10 each). (D) Full trace of Fig. 2f, left panel: CDN calcium signal is depressed upon bath application of 100 µM dopamine (n = 10 each). (E) Whole animal D2R mutants do not show a depression in calcium signaling following bath application of 100 µM dopamine (n = 10 each). (F) Full trace of Fig. 2f, middle panel: animals with the D2R knockdown in the CDNs do not show depression of CDN calcium levels following bath application of 100 µM dopamine (n = 10 each). (G) Animals with the D2R knocked down with a second, independent RNAi in the CDNs do not show depression of CDN calcium levels following bath application of 100 µM dopamine (n = 10). (H) In the absence of ChR2-XXM driven stimulation dopamine application does not alter CaMKII activity in the CDNs (n = 10 each). (I) D2R mutants show a weak increase in CaMKII activity following bath application of 10 µM dopamine (n = 10 each). (J) Animals with the D2R knocked down with a second, independent RNAi in the CDNs show a weak increase in CaMKII activity following bath application of 10 µM dopamine (n = 10 each).

Source data

Extended Data Fig. 6 Satiety sensitizes matings to CDN stimulation.

(A) Electrical silencing of the CDNs counteracts the satiety-induced increase in mating termination in response to a heat threat at 8 mins (n = 16-23). (B) Basal activity of the CDNs does not change with satiety state, as reported by normalizing GCaMP6s to tdTomato fluorescence (n = 10 each). (C) Satiety potentiates the response to a moderate heat threat similarly to moderate CDN activation (n = 22-26). (D) Satiety similarly potentiates the response to a strong heat threat and strong CDN activation (n = 25-34). (E) Satiety and D2R knockdown in the CDNs potentiate the response to direct stimulation of the CDNs similarly (n = 25-33).

Source data

Extended Data Fig. 7 Assessment of dopaminergic signaling in satiated animals.

(A) Dopaminergic stimulation becomes less effective at extending matings after males spend ~2.5 hours mating with females in the satiety assay (n = 18-21). Note: the 2.5 hour time point is reproduced from Fig. 3a. (B) Basal activity of dopamine neurons in the abdominal ganglion does not change with satiety state, as reported by normalizing GCaMP6s to tdTomato fluorescence (n = 6-9). (C) Optogenetically evoked calcium activity in abdominal ganglion dopamine neurons does not change with satiety state (n = 4-6). (D) Recent matings do not change stimulation-induced dopamine received by the CDNs, as reported by GRAB-DA3m (n = 7-8). (E) Full trace of Fig. 3b, left panel: CDN calcium signal does not differ between not satiated and satiated animals (n = 19-20). (F) CDN calcium signal is not significantly depressed by 10 µM DA (n = 10-11). (G) Full trace of Fig. 3b, center left panel: CDN calcium signal is less depressed by bath application of 25 µM dopamine in satiated animals (n = 17-20). (H) Full trace of Fig. 3b, center right panel: CDN calcium signal is less depressed by bath application of 50 µM dopamine in satiated animals (n = 20 each). (I) CDN calcium signal is depressed upon bath application of 100 µM dopamine in not satiated and satiated animals (n = 10 each). (J) Left: schematic of the CaMKII sensor green-Camuiα. Right: Individual traces corresponding to Fig. 3c, left panel: saline application does not increase CaMKII activity in not satiated or satiated flies (n = 10 each). (K) Individual traces corresponding to Fig. 3c, middle panel: bath application of 10 µM dopamine increases CaMKII activity more strongly in not satiated than satiated flies (n = 19-20).

Source data

Extended Data Fig. 8 Activation of Gαo signaling in the CDNs protects matings in real-time.

(A) Knockdown of Gαo in the CDNs does not change copulation duration (n = 14-18). (B) Full trace of Fig. 4c, left panel, CDN calcium signal is depressed upon bath application of 100 µM dopamine in control animals (n = 10 each). (C) Full trace of the data shown in Fig. 4c, middle panel, CDN calcium signal is only slightly depressed upon bath application of 100 µM dopamine in animals with Gαo knocked down in the CDNs (n = 10 each). (D) Similar to controls, males experiencing optogenetic activation of eOPN3 in the CDNs avoid temperatures above 28°C when not mating (n = ~10/group, 3-6 groups per test temperature).

Source data

Extended Data Fig. 9 Knockdown of β-arrestin in the CDNs prevents most aspects of satiety without altering baseline motivation.

(A) Knockdown of β-arrestin (Kurtz) does not change motivational dynamics within a single mating (n = 20-33). (B) Individual traces corresponding to Fig. 5c: dopamine application activates CaMKII in not satiated and satiated animals when Kurtz is knocked down in the CDNs (n = 10 each). (C) Full traces from the data in Fig. 5d (n = 9-10). (D) Knockdown of Kurtz does not block the satiety-induced potentiation of the termination response to optogenetic activation of the CDNs (n = 26-28).

Source data

Extended Data Fig. 10 Scoring extended copulations.

(A) Depiction of flies maintaining normal mating posture throughout a long copulation until natural termination. (B) Depiction of flies displaying stuck behavior, in which the male is physically pulling away from the female but unable to terminate the copulation. (C) Depiction of flies displaying rearing behavior, in which the male temporarily increases the angle between himself and the female without turning to pull away before returning to normal mating posture. Time spent rearing in mating is counted towards overall copulation duration.

Supplementary information

Source data

Source Data Fig. 1 (download XLSX )

Fractions terminating and summary statistics that are used to make graphs.

Source Data Fig. 2 (download XLSX )

Fractions terminating and summary statistics that are used to make graphs in termination experiments.

Source Data Fig. 2 (download JPG )

Image shown in b. ΔF/F or lifetime values for each animal in imaging experiments.

Source Data Fig. 3 (download XLSX )

Individual copulation duration values. ΔF/F or lifetime values for each animal in imaging experiments.

Source Data Fig. 4 (download XLSX )

Fractions terminating and summary statistics that are used to make graphs in termination experiments. ΔF/F or lifetime values for each animal in imaging experiments.

Source Data Fig. 5 (download XLSX )

Fractions terminating and summary statistics that are used to make graphs in termination experiments. ΔF/F or lifetime values for each animal in imaging experiments.

Source Data Extended Data Fig./Table 1 (download XLSX )

Values for percent mating behavior for each group (1 a). Fractions terminating and summary statistics that are used to make graphs in termination experiments. Avoidance indices for each group in the thermosensation assays. Ejaculatory fullness bulb scores.

Source Data Extended Data Fig./Table 2 (download XLSX )

Fractions terminating and summary statistics that are used to make graphs in termination experiments. Individual copulation duration values.

Source Data Extended Data Fig./Table 3 (download XLSX )

Fractions terminating and summary statistics that are used to make graphs in termination experiments. Individual copulation duration values.

Source Data Extended Data Fig./Table 4 (download XLSX )

Fractions terminating and summary statistics that are used to make graphs in termination experiments. Individual copulation duration values.

Source Data Extended Data Fig./Table 5 (download XLSX )

ΔF/F or lifetime values for each animal in imaging experiments.

Source Data Extended Data Fig./Table 6 (download XLSX )

Fractions terminating and summary statistics that are used to make graphs in termination experiments. ΔF/F values for each animal in imaging experiments.

Source Data Extended Data Fig./Table 7 (download XLSX )

Proportions shown in Extended Data Fig. 7. ΔF/F or lifetime values for each animal in imaging experiments.

Source Data Extended Data Fig./Table 8 (download XLSX )

Individual copulation duration values. ΔF/F values for each animal in imaging experiments. Avoidance indices for each group in the thermosensation assays.

Source Data Extended Data Fig./Table 9 (download XLSX )

Fractions terminating and summary statistics that are used to make graphs in termination experiments. ΔF/F values for each animal in imaging experiments.

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Miner, L.E., Gautham, A.K. & Crickmore, M.A. Behavioral devaluation by local resistance to dopamine. Nat Neurosci 28, 2493–2501 (2025). https://doi.org/10.1038/s41593-025-02079-x

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