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Sexual arousal gates visual processing during Drosophila courtship

Nature volume 595, pages 549–553 (2021)Cite this article

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Abstract

Long-lasting internal arousal states motivate and pattern ongoing behaviour, enabling the temporary emergence of innate behavioural programs that serve the needs of an animal, such as fighting, feeding, and mating. However, how internal states shape sensory processing or behaviour remains unclear. In Drosophila, male flies perform a lengthy and elaborate courtship ritual that is triggered by the activation of sexually dimorphic P1 neurons1,2,3,4,5, during which they faithfully follow and sing to a female6,7. Here, by recording from males as they court a virtual ‘female’, we gain insight into how the salience of visual cues is transformed by a male’s internal arousal state to give rise to persistent courtship pursuit. The gain of LC10a visual projection neurons is selectively increased during courtship, enhancing their sensitivity to moving targets. A concise network model indicates that visual signalling through the LC10a circuit, once amplified by P1-mediated arousal, almost fully specifies a male’s tracking of a female. Furthermore, P1 neuron activity correlates with ongoing fluctuations in the intensity of a male’s pursuit to continuously tune the gain of the LC10a pathway. Together, these results reveal how a male’s internal state can dynamically modulate the propagation of visual signals through a high-fidelity visuomotor circuit to guide his moment-to-moment performance of courtship.

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Fig. 1: P1 neurons release and reflect a dynamic state of sexual arousal.
Fig. 2: Modulation of LC10a neurons during courtship.
Fig. 3: P1 neurons represent intensity of courtship and acutely regulate pursuit.
Fig. 4: A network model of LC10a neurons recapitulates male pursuit.

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Data availability

All data underlying this study are available upon request from the corresponding author. Source data are provided with this paper.

Code availability

Code underlying the network model is available at https://github.com/rutalaboratory/LC10NetworkModel.

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Acknowledgements

We thank K. Kuchibhotla, B. Noro, L. Abbott, S. R. Datta, R. Coleman, and P. Brand for comments on the manuscript; I. Ribeiro for discussions and sharing the LC10a split-GAL4 and LC10a-LexA driver lines; Y. Aso for LexAop-GCaMP6s/UAS-CsChrimson flies; J. Weisman and G. Maimon for sharing their designs for an integrated, projector-based system for presenting tethered, walking flies with visual stimuli in advance of publication; R. Coleman, I. Morantte, A. Siliciano, J. Petrillo, and Rockefeller Precision Instrumentation Technologies for technical advice; and G. Maimon, C. Bargmann, L. Abbott, and members of the Ruta laboratory for discussion. Stocks obtained from the Bloomington Drosophila Stock Center, NIH P40OD018537 were used in this study. This work was supported by the Simons Collaboration on the Global Brain, a Reem-Kayden Award, grant 5R35NS111611 from the National Institute of Health to V.R., a David Rockefeller Fellowship and National Science Foundation Graduate Research Fellowship Program to T.H.S., and an HHMI Hanna H. Gray Fellowship to A.O.

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Authors and Affiliations

  1. Laboratory of Neurophysiology and Behavior, The Rockefeller University, New York, NY, USA

    Tom Hindmarsh Sten, Rufei Li, Adriane Otopalik & Vanessa Ruta

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  1. Tom Hindmarsh Sten
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Contributions

T.H.S. and V.R. conceived of and designed the study. T.H.S. and R.L. performed tethered behavioural experiments. A.O. carried out the free courtship assays in Fig. 4. T.H.S. performed and analysed functional imaging experiments. T.H.S. designed and implemented the model. T.H.S. and V.R. analysed data and wrote the manuscript with input from R.L and A.O.

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Correspondence to Vanessa Ruta.

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Extended data figures and tables

Extended Data Fig. 1 A virtual reality preparation for tethered courtship.

a, Schematic of virtual reality preparation. Tethered male flies are placed on an air-cushioned foam ball, whose rotational velocity along all three axes is read out by a single camera via the FicTrac software. During closed-loop experiments, the male’s position in the virtual world is updated on the basis of these rotations, as is the position of the target stimulus on the screen. Changes in the 2D world are mapped to a conical screen and projected by way of a mirror from above. Hardware design by J. Weisman and G. Maimon. b, Schematic of the stimulus presentation during two-photon imaging. Owing to the sterics of the objective, the stimulus is rear-projected onto the screen instead of being projected from above as in a.

Extended Data Fig. 2 Tethered courtship in an open 2D virtual world.

a, Pseudocolour images of a courting male fly during activation of P1 neurons when the visual target is on his left (top), on his right (bottom), or in front of him (middle) showing ipsilateral wing extensions characteristic of courtship song. b, Top, position of the male and autonomously moving fictive female in the 2D world during P1 activation over the course of 200 s. Bottom, histogram of the distance between the male and female target during closed-loop courtship. Note that the male is prevented from bringing the stimulus closer than about 10 mm from his position in the virtual world. c, As in b but for a wild-type male. The increased jitter in the ‘female’ trajectory results from the target frequently reaching the maximum distance from the male and subsequently approaching him along a straight path. d, Top, representative example of the 2D positions of the male and female in a freely courting pair of animals. Bottom, histogram of the distance between the male and female fly. e, Density plot of the relative position of fictive females with respect to the courting male during P1 activation. f, As in e but for a wild-type male. g, Density plot of the location of the female relative to the male in freely courting pairs of animals. Details of statistical analyses and sample sizes are given in Supplementary Table 1.

Source data

Extended Data Fig. 3 The behaviour of aroused animals.

a, Schematic illustrating our definition of the vigour and fidelity of a male’s courtship pursuit. Vigour is quantified as the total turning in the direction of the visual target (normalized within-animal), while fidelity is the correlation between the visual target and the male’s turning. b, Representative example of the vigour, fidelity, and tracking index over the course of a courtship trial. P1 activation is denoted by red line. The male is classified as courting when TI > 0.3, and as disengaged when TI < 0.3 but he remains primed to reinitiate courtship pursuit. c, Comparison of the tracking fidelity, tracking vigour, and tracking index across animals. Each dot represents one frame; black lines indicate zero on axes. d, Distribution of tracking fidelity, tracking vigour, and tracking indices across animals, before (black) and after (red) brief activation of P1 neurons. TI > 0.3 was used as a cut-off to indicate courting males. e, f, Distribution of linear speeds (e) and angular speeds (f) during courtship trials. Lines indicate the thresholds used for denoting animals as ‘moving’ (for example, Fig. 3d, e). g, Distribution of the duration of bouts of courtship (black) and bouts of disengagement (grey) after transient P1 activation. h, Distributions of the angular velocity exhibited by animals that are actively courting (black), that are disengaged (dark grey), or that are passively watching the visual stimulus before P1 activation (light grey). i, As in h but for linear speeds. j, Probability that an animal that is currently courting will transition to disengagement in any given second, plotted over the course of a trial in 10-s bins (red line denotes P1 activation). k, Probability that an animal that is disengaged will transition to courtship in any given second, plotted over the course of a trial in 10-s bins (red line denotes P1 activation). Details of statistical analyses and sample sizes are given in Supplementary Table 1.

Source data

Extended Data Fig. 4 Acute and enduring regulation of courtship arousal.

a, Heading of a courting male before, during, and after the visual target was transiently removed from the screen (30 s). Courtship arousal was induced by a 3-s optogenetic activation of P1 neurons expressing CsChrimson 60 s before stimulus removal. b, Average tracking index of males during trials where stimulus was removed (mean ± s.e.m.). P1 neurons were transiently activated for 3 s, 1 min after the visual target began to oscillate, and target was temporarily removed from the screen for 30 s 1 min after P1 activation. c, Schematic of the preparation allowing male to sample gustatory pheromones to trigger courtship. The male fly was provided with the abdomen of a virgin female to taste with his foreleg while the visual target oscillated on the screen in front of him. d, Example of a male’s heading during a courtship trial, before and after the male tapped the female abdomen with his foreleg (black line indicates tap). Each row consists of three stimulus cycles. e, Pseudocolour image of a male fly sampling the pheromones on a female abdomen. f, Maximal tracking index (right) and duration between the first and last detected bouts of courtship (left) during tapping-induced courtship trials. g, Representative example of male turning during interleaved presentations of either a female target (black line) or a wide-field grating turning in the clockwise (CW, grey) or anticlockwise (counterclockwise; CCW, burgundy) before (left) or during (right) optogenetic activation of P1 neurons. h, Average male turning in response to three cycles of the oscillating female target before (black) or during (red) activation of P1 neurons. i, Average male turning in response to the wide-field grating rotating in the clockwise (grey) or anticlockwise (burgundy) direction before (black) or during (red) activation of P1 neurons. Note that unlike responses to the ‘female’ target in (h), optomotor responses were not enhanced during P1 activation. j, Two-dimensional path of the dynamic visual target used for inducing spontaneous courtship. k, l, Angular position (k) and angular size (l) of the dynamic visual target subtended on the male retina over the course of a 10 min trial. m, Duration between the first and last detected bouts of courtship for trials induced by optogenetic activation of P1 neurons or spontaneously initiated (left), and the maximum tracking fidelity (middle) and vigour (right) displayed by animals in the two conditions. n, Average turning response during courtship in trials where courtship was induced by activation of P1 neurons (left) or spontaneously initiated (right). o, Fraction of males actively engaged in courtship (TI > 0.3) over the course of a 10-min trial in P1-induced trials (left) and spontaneously initiated trials (right). Dashed lines indicate LED onset (red) or the onset of visual motion (right). All shaded line plots are mean ± s.e.m.; *P < 0.05; n.s., P > 0.05; details of statistical analyses and sample sizes are given in Supplementary Table 1.

Source data

Extended Data Fig. 5 P1 neurons are dynamic but strongly correlated to the intensity of courtship pursuit.

a, The average activity of P1 neurons (∆F/F0) plotted against the position of the ‘female’ visual target. Thin grey lines are individual animals, black line is the average across animals. b, Correlation between P1 activity (∆F/F0) and the tracking fidelity, tracking vigour, and tracking index (T.I.) of males. Individual data points are individual animals. c, P1 activity (∆F/F0) plotted against tracking index at the onset of courtship (first 60 s; left) and for the remainder of the trial (right). d, Top, average response of P1 neurons aligned to the onset of courtship. Bottom, average tracking index aligned to the onset of courtship. Note that P1 activity is disproportionally elevated in the first few seconds, indicating that it may reflect additional aspects of the male’s internal state or behaviour that we are not measuring. e, Maximum P1 activity observed across animals as a function of time since courtship initiation. f, Maximum tracking index observed across animals as a function of time since courtship initiation. g, Average correlation between P1 activity (∆F/F0) and the tracking index across animals as a function of time since courtship initiation. All shaded line plots are mean ± s.e.m.; ****P < 0.0001, *P < 0.05; details of statistical analyses and sample sizes are given in Supplementary Table 1.

Source data

Extended Data Fig. 6 P1 neuron activity is uncorrelated with the motor implementation of courtship.

a, Schematic of preparation for evoking optomotor responses using wide-field motion (top), and the turning responses of animals presented with alternating-direction wide-field motion. b, Example of a male’s turning during an optomotor trial. Each row consists of three stimulus cycles. Purple bars indicate when the grating is rotating. c, Example of the functional response (∆F/F0) of P1 neurons during an optomotor trial, before and during periods when the grating turned, as well as the angular velocity and linear speed of the animal. d, Histogram of angular velocities observed during courtship trials (grey) and during optomotor trials (purple). e, Histogram of linear speeds observed during courtship trials (grey) and during optomotor trials (purple). f–i, Scatter plots of P1 activity against the tracking index (f), stimulus position (g), linear speed (h) and angular velocity (i) of all animals during courtship trials. j, Correlation between P1 activity and the parameters explored in f–i during courtship trials. Individual data points are animals. k–n, Scatter plots of P1 activity against the optomotor tracking index (k), velocity of the grating (l), linear speed (m) and angular velocity (n) of all animals during optomotor trials. o, Correlation between P1 activity and the parameters explored in k–n during optomotor trials. Individual data points are animals. All shaded line plots are mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Details of statistical analyses and group sizes are given in Supplementary Table 1.

Source data

Extended Data Fig. 7 LC10a signalling is necessary and amplified during courtship.

a, Schematic of LC10a neurons expressing GtACR1 with approximate ROIs used for silencing (or sham-silencing) in a single hemisphere. b, Average turning of one male to the visual stimulus during silencing of LC10a neurons in the right hemisphere versus sham trials. Note that male fails to execute turns in the direction ipsilateral to silencing. c, Average turning in the directions ipsilateral and contralateral to the hemisphere where LC10a was silenced, compared to sham trials. d, Image of LC10a axon terminals expressing jGCaMP7f in the AOTu. e, Example of functional response (∆F/F0) of LC10a neurons expressing jGCaMP7f during a courtship. Note that, in contrast to recordings made using GCaMP6s (Fig. 2b), calcium transients return to baseline in between responses with this faster indicator. f, Average evoked LC10a responses (∆F/F0) to one stimulus cycle for animals expressing GCaMP6s versus jGCaMP7f. g, Average change in LC10a gain (distance between peak and trough of evoked responses) for animals expressing GCaMP6s versus jGCaMP7f. h, Example of LC10a functional responses during courtship versus during a later period of undirected running with similar linear speed. All shaded line plots are mean ± s.e.m.; n.s., P > 0.05; **P < 0.01. Details of statistical analyses and group sizes are given in Supplementary Table 1.

Source data

Extended Data Fig. 8 LC10a gain can be dissociated from the motor implementation of courtship pursuit.

a, b, Histograms of the linear speeds (a) and angular velocities (b) exhibited by animals in periods classified as courtship versus periods classified as ‘moving’. c, Average evoked LC10a activity (∆F/F0) when the stimulus swept across the ipsilateral hemifield versus the average linear speed of animals in the same time period, colour coded by the average tracking index during the sweep. Red line is the linear fit. d, e, As in c but plotted against the average angular speed (d) or average tracking index (e) exhibited by animals. f, Correlations between LC10a activity and the linear speed, angular speed, and tracking index exhibited by animals. Individual data points denote individual animals. g, Left, schematic of animal being presented with two identical ‘female’ targets, moving in opposition and thus yielding identical stimulation to both eyes. Middle, example of LC10a functional responses plotted against the position of a single target (top) and animal turning responses (bottom). Right: as for middle, but later in the trial when the animal was presented with two opposing targets. P1 neurons were activated continuously. Note that LC10a neurons responded even when the male failed to turn ipsilaterally when two targets were present. h, Average LC10a activity during presentation of two opposing visual targets (top). i, Left, average evoked LC10a activity during ipsilateral sweeps of the visual target versus the total turning exhibited in the direction of the visual target during the same period. Right, average evoked LC10a activity during ipsilateral sweeps of either of the two visual targets versus the total turning exhibited in the direction of the visual target during the same period. j, Correlation between LC10a evoked responses and ipsilateral turning. Individual data points denote individual animals. k, Average peak-normalized responses (∆F/F0) of LC10b/c neurons during courtship versus during locomotion. l, As in k but for LC10d neurons. m, n, Average evoked LC10a functional response (∆F/F0, k) and average evoked ipsiversive turning (n) as a function of the average angular size of the visual target on each stimulus cycle. All shaded line plots are mean ± s.e.m.; n.s., P > 0.05; *P < 0.05, **P < 0.01, ***P < 0.001. Details of statistical analyses and group sizes are given in Supplementary Table 1.

Source data

Extended Data Fig. 9 LC10a neurons exhibit sparse and selective connectivity in the central brain.

a, Examples of identified LC10a, LC10b, LC10c, and LC10d neurons in the female hemi-brain connectome23. b, Morphology of all identified LC10a-d neurons (n = 248). c, Correlation matrix of the outputs from all LC10 neurons, sorted by their assigned subtype. Note that individual subtypes have strongly correlated outputs that are largely distinct from the output patterns of other subtypes. d, t-SNE plot of the output connectivity matrix of all identified LC10 neurons, labelled according to the manually assigned subtype. The output connectivity naturally segregates LC10 neurons into four groups. e, f, Same as c, d but based on the input connections to LC10 neurons in the AOTu. g, Morphology of all non-visual output neurons from LC10a neurons with at least 10 synaptic connections, grouped by projections to the LALs (left) versus to the inferior bridge (IB; right). h, As in g but for non-visual input neurons to LC10a neurons in the AOTu. i, Representative example of trans-synaptic tracing of LC10a neurons in the male using trans-Tango24. Magenta denotes labelled LC10a neurons, and cyan the labelled postsynaptic partners. Similar results were obtained across four male brains. j, Histogram of synaptic weights between all LC10a neurons and their postsynaptic partners. k, Number of input and output synapses to and from LC10a neurons from the 10 most common brain regions (superior intermediate protocerebrum (SIP), lateral accessory lobe (LAL), superior medial protocerebrum (SMP), inferior bridge (IB), superior posterior slope (SPS), posterior ventrolateral protocerebrum (PVLP), posteriolateral protocerebrum (PLP), superior medial protocerebrum (SMP), wedge (WED)). R and L indicate the right and left hemisphere, respectively.

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Extended Data Fig. 10 P1 neurons enhance the gain of LC10a neurons.

a, Left, schematic of synchronous recordings from P1 neurons in the LPC and LC10a neurons in the AOTu. Middle, cross-covariance of P1 neuron activity and LC10a activity during spontaneous courtship trial. Right, as for middle, but zoomed in to highlight that P1 neuron activity leads LC10a activity. Maximum covariance occurred at lag of −500 ms. b, LC10a responses to presentation of a 10° sweeping dot in the progressive or regressive direction before and during activation of P1 neurons. Top, average LC10a response during presentation of a regressively (orange) or progressively (blue) moving stimulus in the absence of P1 activation. Bottom, average LC10a response during presentation of a regressively (orange) or progressively (blue) moving stimulus in the presence of P1 activation. c–e, As in b but for a sweeping 25° sphere (c), a sweeping 10° wide tall bar (d), or an approaching sphere expanding from 10° at 20°/s (e). Red indicates P1stimulation and black indicates pre-stimulation baseline throughout. f, Response modulation index (see Methods) for each stimulus presented before and during P1 activation, indicating that responses to the distinct visual stimuli are near-uniformly enhanced. g, Average evoked ipsilateral turning in response to progressive motion of the different targets during P1 activation, plotted against the average evoked LC10a response in the same period. Note that turning responses evoked by the motion of these diverse stimuli were proportional to the magnitude of LC10a evoked calcium transients: sweeping dots evoked the strongest turns, bars evoked much weaker turns, and slowly looming spheres did not elicit any turning on average, presumably because both eyes were stimulated equally. h, Average evoked linear speed in response to progressive motion of the different targets during P1 activation, plotted against the average evoked LC10a response in the same period. i, Direction selectivity index (see Methods) for sweeping stimuli presented during baseline recordings or during continuous P1 activation. Positive values indicate a preference for progressive motion, negative values indicate preference for regressive motion. All shaded line plots are mean ± s.e.m.; n.s., P > 0.05. Details of statistical analyses and sample sizes are given in Supplementary Table 1.

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Extended Data Fig. 11 Motion-direction selectivity during courtship pursuit.

a, Left, example image of LC10a–LexA axon terminals in the AOTu, with 48 ROIs of strongly correlated pixels automatically selected using the CaImAn-CNMF framework48 overlayed. Right, as for left, but with ROIs colour-coded according to their exhibited direction selectivity index (positive values indicate a preference for progressive motion, negative values indicate preference for regressive motion). b, Heat map of the average evoked responses to progressive (right) and regressive (left) sweeps of the 25° sphere during P1 activation for the 48 ROIs shown in a. Each row represents the average evoked fluorescence across 10 trials for each ROI. c, Average evoked responses to a progressively versus regressively moving 25° dot across all ROIs from all animals (300 ROIs across 7 males). d, Direction selectivity index for all ROIs across animals (see Methods). Black line denotes zero; positive values indicate a selectivity for progressive motion, negative values indicate a selectivity for regressive motion. e, Top, schematic of monocular stimulation. ‘Female’ targets were presented to one eye alone, and moved in either the regressive or progressive direction with respect to that eye with a 5-s ISI. Bottom, average turning of males in response to monocular stimuli moving regressively (left) or progressively (right). f, Turning responses of LC10a circuit model without motion-direction selectivity, with regressive-motion selectivity, and with progressive-motion selectivity. g, Left, normalized LC10a receptive fields with varying rise-times (κ, see Methods). Right, correlation between predicted and actual responses to the simple stimulus in f for the receptive fields shown to the left. h, From left: average turning response to a single stimulus cycle; predicted response from full model to a single stimulus cycle; predicted response of a model with no binocular overlap; predicted response of a model not selective for progressive versus regressive motion. i, Example of predicted versus actual turning response to two targets with a drifting phase-offset (as in Fig. 4d) across the courtship trial. Black line indicates when first target is present, grey line indicates when the second target is present. j, Left, average correlation between stimulus 1 and predicted turning (cyan) during dual dot presentations. Right, average correlation between stimulus 1 and the turning of males during dual dot presentations. In grey is what the correlation to stimulus 1 would be if the animal perfectly tracked stimulus 2. Positive x-values indicate that the first stimulus leads in phase. All shaded line plots are mean ± s.e.m.; **P < 0.01, ****P < 0.0001. Details of statistical analyses and sample sizes are given in Supplementary Table 1.

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Extended Data Fig. 12 Network model predicts turning dynamics of freely courting males.

a, Examples of predicted versus actual turning of freely courting males over the first 100 s of courtship. b, Frame-by-frame predicted versus actual male turning over the course of the full courtship trials for the pairs shown in a (5–10 min); red line shows the linear fit. Details of statistical analyses and sample sizes are given in Supplementary Table 1.

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Extended Data Fig. 13 Incorporating P1 neural activity improves model performance.

a, Hit rate (fraction of predicted turns accompanied by a real turn; true positive rate) and false-alarm rate (fraction of predicted turns not accompanied by a real turn; false positive rate) of the naive model (lacking P1 input) versus when input current to LC10a neurons is scaled by the functional responses of P1 neurons. b, Example of the predicted turning over a courtship trial by a model lacking P1 input (as in Fig. 4a) in which input current to LC10a neurons is consistently high. c, d, Two examples of actual (left) versus predicted (middle) turning responses when the activity of P1 neurons (right) is incorporated into the model. Compare to model without P1 input in b. Black lines indicate when stimulus is oscillating. Details of statistical analyses and sample sizes are given in Supplementary Table 1.

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Supplementary Tables

This file contains Supplementary Tables 1-2.

Reporting Summary

Video 1

Tethered males court a fictive female target. Representative example of a male courting a virtual ‘female’ target in closed loop during continuous optogenetic activation of P1 neurons. Note frequent unilateral wing-extensions, indicating production of courtship song.

Video 2

Males effectively purse the female target in closed-loop. Representative example of the visual stimulus presented to a male courting a virtual ‘female’ target in closed loop during continuous optogenetic activation of P1 neurons. Female moves autonomously from the male in the virtual world. Note that the male efficiently centers the ‘female’ target in his field-of-view, and actively brings it closer to him in the virtual world.

Video 3

Transient activation of P1 neurons drives sustained courtship. Representative example of a male presented with a simple, repeating visual target before and after P1 neurons are optogenetically activated. Blue line indicates the integrated path of the male. Note the stimulus can be observed as a dark target in the background, and that the male readily exhibits unilateral wing-extensions in the direction ipsilateral to the target after P1 neurons have been activated.

Video 4

A dynamic visual target for evoking spontaneous courtship. Example of the translating visual stimulus used to drive males to spontaneously initiate courtship. The target traverses a steady arc but appears to advance and recede.

Video 5

Males spontaneously initiate courtship towards a dynamic visual target. Representative example of a male spontaneously initiating courtship towards the translating visual stimulus shown in Supplementary Video 4, with his trajectory and unilateral wing extensions shown. Note that the male does not exhibit any structured turning before the visual stimulus is presented, and the performance of alternating unilateral wing-extensions, early during the courtship bout. Faithful tracking persists after the male stops performing wing-extensions.

Video 6

Two visual targets with a drifting phase relationship. Example of the two-target stimulus presented to males, with one target moving slightly slower than the other, causing the phase-relationship between the two to drift over time. 2x playback.

Video 7

An LC10a network model predicts the behaviour of freely courting males. Three examples of the actual versus predicted paths of how a male pursues a female target in two dimensions. Pink dot is the trajectory of the real female fly; blue dot is the trajectory of the real male fly; green is the trajectory of a simulated fly. The simulated fly is initialized at the same position as the real male fly and matches its linear velocity to that of the real female fly.

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Hindmarsh Sten, T., Li, R., Otopalik, A. et al. Sexual arousal gates visual processing during Drosophila courtship. Nature 595, 549–553 (2021). https://doi.org/10.1038/s41586-021-03714-w

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