Fly husbandry and genotypes

Flies have been cultivated on a cornmeal, molasses and yeast medium below a 12 h–12 h gentle–darkish cycle at 25 °C and 60% humidity. All the experiments have been carried out on feminine flies bearing at the least one wild-type allele of the white gene. The experimenters weren’t blinded.

Drosophila melanogaster of the next genotypes have been used to focus on transgene expression to the respective varieties of neuron: P{R48A07-p65.AD}attP40, P{10XUAS-IVS-mCD8::GFP}su(Hw)attP5; P{VT046779-GAL4.DBD}attP2 was used to label Mi9 neurons, P{R13E12-p65.AD}attP40/+; P{R59C10-GAL4.DBD}attP2/P{40XUAS-IVS-mCD8::GFP}attP2 was used to label Tm3 neurons, P{R19F01-p65.AD}attP40/+; P{R71D01-GAL4.DBD}attP2/P{40XUAS-IVS-mCD8::GFP}attP2 was used to label Mi1 neurons, P{R48A07-p65.AD}attP40, P{10XUAS-IVS-mCD8::GFP}su(Hw)attP5; P{R13F11-GAL4.DBD}attP2 was used to label Mi4 neurons, P{R26H02-p65.AD}attP40/+; P{R29G11-GAL4.DBD}attP2/P{40XUAS-IVS-mCD8::GFP}attP2 was used to label C3 neurons and P{R42F06-p65.AD}attP40, P{10XUAS-IVS-mCD8::GFP}su(Hw)attP5; P{VT037588-GAL4.DBD}attP2 (abbreviated T4 > GFP) was used to label T4 neurons, with a choice for subtypes T4c and T4d^17,27,45,46. In electrophysiological experiments, P{TRiP.HMC03585}attP40/P{R42F06-p65.AD}attP40, P{10XUAS-IVS-mCD8::GFP}su(Hw)attP5; P{VT037588-GAL4.DBD}attP2/+ (abbreviated T4 > GluClα^RNAi) and P{TRiP.HMS02199}attP2/P{R42F06-p65.AD}attP40, P{10XUAS-IVS-mCD8::GFP}su(Hw)attP5; P{VT037588-GAL4.DBD}attP2/+ (abbreviated T4 > Nmdar1^RNAi) have been used to silence the expression of GluClα and Nmdar1, respectively⁴⁷.

In behavioural experiments, P{UAS-Dcr-2.D}2; P{R39H12-GAL4}attP2 (abbreviated T4/T5 >), which yields robust and complete expression in T4 and T5 neurons, was used to drive both P{TRiP.HMC03585}attP40 (abbreviated GluClα^RNAi) or P{TRiP.HMS02199}attP2 (abbreviated Nmdar1^RNAi). For the experiments in Prolonged Information Fig. 10c–f, P{R59E08-p65.AD}attP40; P{R42F06-GAL4.DBD}attP2 was used as the motive force line. All flies, together with the parental controls, have been heterozygous for the respective transgenes. P{UAS-Dcr-2.D}2/P{10XUAS-IVS-mCD8::GFP}su(Hw)attP5; P{R39H12-GAL4}attP2/+ and P{R59E08-p65.AD}attP40/P{10XUAS-IVS-mCD8::GFP}su(Hw)attP5; P{R42F06-GAL4.DBD}attP2/+ have been used to visualise the expression sample of the respective driver traces immunohistochemically.

Apart from the pressure used to label C3 (a present from A. Nern and M. Reiser), the entire flies have been obtained from the Bloomington Drosophila Inventory Middle.

Histology

Brains of feminine flies (aged 1–3 days) have been dissected in phosphate-buffered saline (PBS; 137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.3) and stuck in 4% (w/v) paraformaldehyde in PBS in a single day at 4 °C, adopted by 4 30 min washes in PBS containing 0.2% (v/v) Triton X-100 (PBT). To label biocytin-filled neurons, the samples have been incubated with DyLight 633-conjugated streptavidin (21844, Invitrogen, 1:200) for 48 h at 4 °C, adopted by 4 30 min washes in PBT. To visualise GFP expression patterns pushed by R39H12-GAL4 and R59E08-AD; R42F06-DBD, brains have been fastened for 25 min at room temperature and blocked in PBT containing 10% regular goat serum (NGS) in a single day at 4 °C. Synaptic constructions and GFP have been labelled, first with mouse anti-bruchpilot antibodies (nc82, AB2314866, Developmental Research Hybridoma Financial institution, 1:20) and hen anti-GFP antibodies (600-901-215S, Rockland, 1:400), respectively, for 48 h after which with Atto 647N-conjugated goat anti-mouse IgG antibodies (610-156-040, Rockland, 1:300) and Alexa 488-conjugated goat anti-chicken IgY antibodies (A-11039, Invitrogen, 1:500), respectively, for 72 h, each diluted in PBT containing 5% NGS, at 4 °C. Immunodecorated samples have been mounted in Vectashield antifade mounting medium (Vector Laboratories) and imaged on a Leica TCS SP8 confocal microscope outfitted with an HCX PL APO ×63/1.30 NA glycerol-immersion goal (506353, Leica). Micrographs have been acquired utilizing the Leica Software Suite X (Leica) and processed utilizing the Fiji distribution of ImageJ (v.2.0)⁴⁸.

Patch-clamp recordings

For whole-cell recordings in vivo^49,50, feminine flies aged 2–24 h post-eclosion have been cold-anaesthetized and stuck to a {custom}, laser-cut polyoxymethylene mount with mushy thermoplastic wax (Agar Scientific). The preparation was submerged in extracellular answer (pH 7.3) containing 5 mM TES, 103 mM NaCl, 3 mM KCl, 26 mM NaHCO₃, 1 mM NaH₂PO₄, 1.5 mM CaCl₂, 4 mM MgCl₂, 10 mM trehalose, 10 mM glucose and seven mM sucrose (280 mOsM, equilibrated with 5% CO₂ and 95% O₂). Cuticle, adipose tissue and trachea have been surgically eliminated in a window giant sufficient to reveal the left dorsal optic lobe. Patch pipettes (15–20 MΩ) have been fabricated from borosilicate glass capillaries with outer and inside diameters of 1.5 mm and 1.17 mm or 0.86 mm, respectively, utilizing a P-97 (Sutter Devices) or a PC-10 (Narishige) micropipette puller. Pipettes have been polished utilizing a microforge (MF-830, Narishige) and stuffed with answer (pH 7.3) containing 10 mM HEPES, 140 mM potassium aspartate, 1 mM KCl, 4 mM MgATP, 0.5 mM Na₃GTP, 1 mM EGTA and 10 mM biocytin (265 mOsM). Inexperienced fluorescent somata have been focused visually utilizing a mixture of bright-field and epifluorescence microscopy on an InVivo SliceScope (Scientifica) or an Axio Scope.A1 microscope (Zeiss), every outfitted with a ×60/1.0 NA water-immersion goal (LUMPLFLN60XW, Olympus) and an LQ-HXP 120 gentle supply (Leistungselektronik Jena). Transillumination was achieved by butt-coupling a white LED (MCWHD5, Thorlabs) to a liquid gentle information, the far finish of which was positioned caudally at a distance of 1 cm to the fly permitting for an unobstructed discipline of view. To achieve entry to cell membranes, a micropipette was used to make a small incision within the perineural sheath. Alerts have been recorded at room temperature (21–23 °C) with a MultiClamp 700B amplifier, low-pass-filtered and sampled at 10 kHz utilizing a Digidata 1550B digitizer managed via pCLAMP 11 software program (all from Molecular Gadgets). Information have been corrected for the liquid junction potential and analysed utilizing custom-written software program in Python v.3.7 (Python Software program Basis) utilizing NumPy v.1.15, Pandas v.0.25, SciPy v.1.3, Matplotlib v.3.0 and pyABF v.2.1 (https://pypi.org/challenge/pyabf/). After temporal alignment, current-clamp knowledge have been analysed at a sampling charge of 1 kHz. Probably the most adverse membrane potential recorded inside 2 min after break-in, in darkness and within the absence of a holding present was taken to symbolize the resting potential. Solely cells with a measured resting potential that was extra adverse than −25 mV have been characterised additional. Enter resistances, as plotted in Fig. 2, have been calculated on the idea of linear matches to the steady-state voltage modifications elicited by 1 s steps of hyperpolarizing currents (2 pA increments, beginning at −10 pA). In voltage-clamp recordings, voltage steps have been utilized 2 s prematurely of pharmacological functions and linear leak currents have been subtracted.

Visible stimulation in electrophysiological experiments

Visible stimuli have been projected with two mirrors onto a cylindrical display screen utilizing two DLP Lightcrafter 3000 pico projectors (Texas Devices) as beforehand described²⁰. The display screen coated 180° in azimuth and 105° in elevation of the fly’s left frontal visible discipline and doubled as a Faraday defend. Proscribing the projectors to the inexperienced channel (500–600 nm) allowed for a refresh charge of 180 Hz at 8-bit color depth and a maximal luminance of 1,274 cd m⁻². The common luminance of stimuli, which have been introduced in full distinction, was set to an 8-bit greyscale worth of 128 equivalent to a median luminance of ~637 cd m⁻². Stimuli have been created and predistorted to account for the curvature of the display screen utilizing the Panda3D sport engine in Python v.2.7.

Receptive fields have been positioned and characterised utilizing a binary white-noise stimulus with a pixel measurement of two.8° × 2.8°. Samples have been drawn at a charge of 60 Hz and projected onto the display screen for durations starting from 3 min to twenty min. Stimuli and concurrently recorded membrane potentials have been time-locked utilizing a repeatedly recorded set off sign on the display screen. Stimulus information have been exported after lossless compression and cross-correlated to every neuron’s recorded membrane voltage utilizing customary methods for reverse correlation in Python (v.3.7)²⁰. Sluggish voltage drifts have been corrected put up hoc by subtracting a low-pass-filtered model of the sign obtained utilizing a Gaussian blur with a regular deviation of 60 s. The reverse correlation was calculated as

the place V_m denotes the neuron’s baseline-subtracted membrane voltage at time level t and S denotes the stimulus at place x and time level t − τ for values of τ starting from −0.5 to +3.0 s. The ensuing spatiotemporal receptive fields have been transformed into customary scores. Solely neurons with clear customary rating peaks (usually >4 s.d. from the imply) and with receptive discipline centres >8 px (22.48°) from the bezel of the display screen have been included within the evaluation to ensure full protection of the encompass. Receptive fields have been normalized and aligned in house utilizing the extremum (that’s, the utmost or minimal with the very best absolute worth) of the usual rating as some extent of reference, which was positioned at 0°. After cropping the person spatial receptive fields to the biggest frequent area holding knowledge from all neurons, scores have been averaged throughout neurons of 1 class. For Fig. 1, averages have been upsampled by an element of 10 by linear interpolation and smoothed with a Gaussian filter (1.8 px s.d.). For direction-selective T4 neurons, particular person receptive fields have been rotated in house to align alongside the neurons’ PDs; due to this fact, in Fig. 1e, azimuth and elevation don’t essentially correspond to horizontal and vertical coordinates on the display screen, however to coordinates parallel and orthogonal to the T4 cell’s PD.

To find out a neuron’s PD, square-wave gratings with a spatial wavelength of 30° spanning the total extent of the display screen have been moved at a temporal frequency of 1 Hz in eight completely different instructions separated by 45°. The neuron’s peak membrane voltage throughout movement, after subtracting a 1 s prestimulus baseline, was taken to symbolize the magnitude of a Euclidean vector v(φ) pointing within the route given by the angle of rotation φ of the related stimulus. PD was outlined because the route of the resultant of all particular person vectors. Temporal frequency tuning curves have been measured utilizing gratings of the above properties that have been moved alternatingly in PD and ND (that’s, PD + 180 ) at temporal frequencies starting from 0.5 Hz to 16.0 Hz. ΔV_m was outlined as absolutely the distinction between the maximal and minimal membrane potential.

The fine-grained directional tuning curves in Fig. 5 have been assessed utilizing ON edges transferring at 30° s⁻¹ in 36 evenly spaced instructions. Membrane potentials have been recorded within the presence of a continuing holding present of −1 pA, which enabled steady recordings over prolonged intervals of time. In Fig. 5c, |v(φ)| was outlined as the utmost of a Voigt profile match to the membrane potential in a 700 ms time window surrounding the height response throughout movement within the respective route utilizing the VoigtModel operate of the lmfit.fashions module in Python v.3.7. Thus the readout included extra knowledge factors than simply the maxima of the uncooked traces. To make directional tuning curves comparable between experiments and genotypes, every neuron’s PD was aligned put up hoc to 0° and its tuning curve was minimal–most normalized. Directional tuning was quantified because the magnitude of the resultant vector divided by the sum of the person vectors’ magnitudes:

For the experiments in Fig. 3, vivid (ON) and darkish (OFF) edges have been moved throughout the display screen at a velocity of 30° s⁻¹. The responses of particular person neurons of 1 sort have been temporally aligned primarily based on the cross-correlation most between the time spinoff of the low-pass-filtered membrane potential of every neuron and that of 1 hand-picked template neuron in response to ON edges (transferring in PD for T4 cells). The responses of various enter neuron courses have been aligned primarily based on the relative distances of the template neurons’ receptive discipline centres on the display screen. Appropriate alignment was verified by recording gentle intensities from a 5°-wide space of the display screen positioned on the respective template neuron’s receptive discipline centre utilizing a custom-built photodiode below similar stimulus situations.

Time-locked measurements of a T4 neuron’s membrane potential and enter resistance (Fig. 4 and Prolonged Information Fig. 9) have been achieved via repeated shows of similar stimuli with various holding present amplitudes starting from −5 to 0 pA. The slope of a linear regression of voltages onto holding currents offered a measure of the neuron’s enter resistance at every time level. For experiments with solely two completely different holding present amplitudes, the slope of the regression is equal to the enter resistance calculated as ΔV_m/ΔI, the place ΔV_m denotes the change in membrane potential and ΔI denotes the change in holding present in between repetitions. Resistances proven in Fig. 4 have been smoothed with a Gaussian filter (13 ms s.d.). Enter resistances didn’t change considerably all through recording periods. The distinction in enter resistance between the beginning and the tip of recording periods averaged at 0.28 ± 0.56 GΩ (imply ± s.e.m., n = 30 cells; P = 0.6143, two-tailed paired Pupil’s t-test).

Pharmacology

For functions of glutamate, acetylcholine and GABA, a micropipette with a bore diameter of 5 µm was stuffed with 1 mM of neurotransmitter (dissolved in extracellular answer) and aimed on the GFP-labelled T4 dendrites in layer 10 of the medulla. To elicit transient neurotransmitter responses in patch-clamped T4 neurons, strain (50 kPa) was utilized in 100 ms pulses utilizing a PDES-02DX pneumatic drug ejection system (NPI Digital). For long-lasting responses throughout enter resistance measurements, pulse instances have been elevated to 500 ms. Two wild-type neurons have been misplaced after the third glutamate utility throughout patch-clamp recordings for Fig. 2e and have been excluded from the repeated-measures evaluation.

Multi-compartment mannequin

We constructed a passive compartmental mannequin of a T4 neuron (Prolonged Information Fig. 4c, d) in Python v.3.7 to account for attainable space-clamp issues as a consequence of neuronal morphology in voltage-clamp experiments and to evaluate sign propagation between dendrite and soma (Prolonged Information Fig. 4e, f). The mannequin was primarily based on an electron microscopic reconstruction⁷ (http://neuromorpho.org/neuron_info.jsp?neuron_name=T4a-25_85) and comprised 2,012 compartments. A connectivity matrix, which held values of 1 the place two compartments have been related and values of 0 in any other case, was used as a template to calculate a conductance matrix M. The latter was primarily based on the three-dimensional coordinates and the size in addition to the diameter of every compartment assuming, until said in any other case, an axial resistivity (R_a) of 150 Ω cm, a membrane resistance (R_m) of 28 kΩ cm², and a selected membrane capacitance (C_m) of 1 µF cm⁻². All parameters have been on the identical scale as these generally used to mannequin Drosophila neurons⁵¹ and have been thought of to be uniform throughout your entire cell. Various R_a and R_m over a biophysically believable vary had negligible results on mannequin output (Prolonged Information Fig. 4f, g).

The voltage vector V_m(t) indicating the membrane potential of every compartment and at every time level t was decided by utilizing the sparse.linalg.spsolve operate of the SciPy v.1.3 module to iteratively resolve the matrix equation M × V_m(t) = V_m(t − 1) × c_m/Δt + E_leak × g_leak + I(t), the place V_m(t − 1) denotes the voltage vector on the earlier time level, c_m is the vector holding the precise capacitances of all compartments, Δt denotes the time step, E_leak denotes the leak reversal potential, g_leak denotes the vector holding the precise transmembrane leak conductances of all compartments and I(t) is the vector indicating the present injected at time level t into every compartment. Simulations have been carried out with a set Δt of 0.1 ms. If solely steady-state was thought of, the diagonal of the conductance matrix M held no capacitive conductances and the best facet of the equation simplified to E_leak × g_leak + I(t). On the time of transmitter utility, synaptic conductances have been added each to the diagonal of the conductance matrix and, multiplied by the reversal potential of the present, to the best facet of the equation.

To simulate voltage clamp, the present injected on the soma was calculated on the idea of the distinction between the chosen command voltage V_cmd and the precise potential on the soma V_m,soma utilizing a proportional-integral management loop that served to emulate a voltage-clamp amplifier in Python v.3.7. The present to be injected at time level t was calculated as I(t) = Ok_p × (V_cmd(t) − V_m,soma(t)) + Ok_i× I(t − 1); the place Ok_p denotes the proportional acquire and Ok_i the integral acquire. With values of two × 10⁹ and 1 for Ok_p and Ok_i, respectively, V_m,soma may very well be clamped reliably on the desired V_cmd below all situations and synaptic inputs.

Single-compartment mannequin

Recorded membrane voltages of enter neurons have been averaged, minimal–most normalized (retaining the sign ratios throughout stimuli) and transformed into relative conductances utilizing a rectilinear switch operate with two free parameters per neuron: a threshold beneath which all conductances have been set to 0 and a acquire (that’s, a scaling issue). Making an allowance for a median inter-ommatidial angle θ of 4.8° (refs. ^52,53) and the sting velocity v of 30° s⁻¹, conductances of Mi9 neurons and people of Mi4 and C3 neurons have been superior or delayed in time, respectively, by Δt relative to these of Mi1 and Tm3 neurons, relying on the angle φ of the digital edge: Δt = θ cosφ/v.

For every stimulus situation, the membrane potential of the T4 neuron was calculated as

the place g denotes the relative conductance related to every enter neuron and E denotes the reversal potential of the respective synaptic present with E_Glu = −71 mV, E_ACh = −21 mV and E_GABA = −68 mV as measured/modelled in voltage-clamp experiments (Prolonged Information Fig. 4a–d). Owing to the compact measurement of a T4 neuron, the small amplitudes of capacitive currents (in relation to the steady-state amplitudes) and their quick time constants (in relation to these of synaptic currents) eradicated the necessity for a differential equation to calculate V_m. Free parameters (thresholds, positive factors, E_leak and g_leak) have been estimated from a least-squares match to measured membrane voltage traces of T4 neurons, computed with the assistance of the optimize.decrease operate of the SciPy v.1.3 module and hand-tuned utilizing a FaderPort 16-channel combine manufacturing controller (Presonus). Higher and decrease bounds for parameter values have been set to 0 and 1 for thresholds, 0 and a couple of for positive factors, −80 mV and −45 mV for E_leak, and 0 and three for g_leak, respectively. The parameters used for the simulations proven in Figs. 3b, c and 5a and Prolonged Information Figs. 7b, c and eight have been as follows: Mi9_acquire = 0.92, Tm3_acquire = 0.35, Mi1_acquire = 0.65, Mi4_acquire = 1.10, C3_acquire = 1.49, Mi9_thld = 0.20, Tm3_thld = 0.35, Mi1_thld = 0.88, Mi4_thld = 0.44, C3_thld = 0.70, E_leak = −65.0 mV and g_leak = 0.50, the place ‘thld’ refers back to the respective threshold values.

To validate our alternative of parameters and to quantify the sensitivity, robustness and uniqueness of parameter units, we resorted to simulation-based inference²⁹, which enabled us to look at the total vary of attainable parameter mixtures. We used 20,000 mannequin simulations, drawing parameters from uniform distributions throughout the above bounds, to coach the factitious neural community applied within the sequential neural posterior estimation (SNPE) algorithm of the software program package deal sbi (v.0.8)⁵⁴. On the idea of Bayesian inference, SNPE offered a conditional likelihood distribution P(α|V_knowledge), which is excessive for parameter units α which might be according to the experimentally measured voltage traces V_knowledge, however near zero in any other case. To visualise P(α|V_knowledge) we drew 10,000 pattern parameter units which might be suitable with V_knowledge and in contrast them to our chosen parameters (Prolonged Information Fig. 6). All the simulations have been written in Python v.3.7.

Behaviour

Feminine flies (aged 1–5 days) have been cold-immobilized and connected to a pin with light-curing composite glue (Sinfony Opaque Dentin, 3M) utilizing dental curing gentle (440 nm, New Woodpecker). 5 impartial locomotion recorders³² have been operated in parallel. In every recorder, a tethered fly was positioned on high of an air-suspended polyurethane sphere with a diameter of 6 mm and a weight of round 40 mg. The sphere floated freely on an air stream provided by a rotary vane pump (G6/01-Ok-EB9L, Gardner Denver Thomas) via an inlet on the backside of a concave holder, permitting the strolling fly to rotate the sphere about any axis via its centre. The rotation of the spherical treadmill, lit by an infrared LED (JET-800-10, Roithner Electronics), was tracked at 4 kHz and digitized at 200 Hz utilizing a custom-designed system primarily based on two optical pc mouse sensors centered on two 1 mm² equatorial squares at ±30° from the centre of the sphere⁵⁵. A digital camera (GRAS-20S4M-C, Level Gray Analysis) was used to facilitate correct positioning of the fly on the ball. To encourage extended strolling, the air temperature surrounding the fly was maintained at 34 ± 0.1 °C utilizing a custom-built air con system with a Peltier heater (QC-127-1.4-6.0MS, Fast-Cool) and a thermometer positioned beneath the sphere.

Visible stimuli have been introduced with a refresh charge of 120 Hz on three liquid crystal shows (2233RZ, Samsung) organized vertically to type a U-shaped visible area surrounding the fly, which spanned roughly 270° in azimuth and 120° in elevation of the fly’s visible discipline at a decision of <0.1°. The maximal luminance of the shows was 131 cd m⁻²; the typical depth of stimuli, which have been introduced at a Michelson distinction of fifty%, was set to an 8-bit greyscale worth of 100. Stimuli have been created, and predistorted to imitate a cylindrical panorama, utilizing the Panda3D sport engine in Python v.2.7.

In open-loop experiments, ON and OFF edges have been moved at a velocity of 60° s⁻¹ in 16 evenly spaced instructions. Owing to the geometry of the visible area, full translation of edges at completely different angles required variable quantities of time. Thus, to restrict stimulus durations to five s, an fringe of which the route of movement deviated from the cardinal instructions was initialized with a small section of the sting already current in one of many outer corners (by no means overlaying any a part of the central show). Edges began transferring 0.5 s after stimulus initialization and crossed the sector inside 5 s. In a single experiment (~80 min), flies skilled 50 trials of both ON or OFF edges transferring in all 16 instructions in a pseudorandom order. The primary 15 trials have been used to equilibrate the temperature and to accustom the fly to the treadmill and have been excluded from analyses. As inclusion standards, we used a ahead strolling pace of ≥0.15 cm s⁻¹ on a trial-by-trial foundation and a minimal of ten trials per fly. To right for a attainable fixed turning bias, the time-averaged rotational velocity of every full trial (comprising all 16 instructions) was subtracted from all measurements of the corresponding trial. The optomotor response was quantified as the typical rotational velocity throughout 5 s of edge movement within the corresponding route. The slope of a linear regression of optomotor responses onto absolutely the horizontal stimulus parts |cosφ| served as a single measure of an animal’s angular velocity throughout completely different edge angles φ.

In closed-loop experiments, bar-fixation was assessed utilizing a ten°-wide darkish vertical bar, the place of which alongside the azimuth was managed in actual time by the rotation of the spherical treadmill (Δbar place = −rotation about z axis, up to date each ~9 ms). The bar appeared at a random place between −180° and 180° at first of every 20 s trial, throughout which the fly may management the bar’s place via its strolling behaviour. One experiment (~60 min) consisted of 180 trials, the primary 40 of which weren’t analysed, as they served to equilibrate the temperature and to accustom the fly to the digital setting. For the outcomes introduced in Prolonged Information Fig. 10d–f, every experiment consisted of 80 longer multi-stimulus trials, the primary 10 of which have been excluded. Solely trials with a ahead strolling velocity of ≥0.40 cm s⁻¹ and flies with at the least 50 (20 for Prolonged Information Fig. 10d–f) of such trials have been included within the evaluation. To keep away from attainable turning bias (for instance, as a consequence of skewed mounting), flies whose common turning deviated from zero by >10° s⁻¹ have been excluded. Likelihood density features of bar positions have been calculated for every 20 s trial utilizing a bin width of 5° earlier than averaging over trials. The measure ‘fixation in entrance’ was obtained by summing the chances of discovering the bar in a 60° window in entrance of the fly and averaging these possibilities over trials.

Statistics and reproducibility

Statistical assessments have been carried out in Prism v.9.2 (GraphPad). Particulars, together with check statistics, levels of freedom and actual P values for statistical analyses of information proven in Figs. 2 and 5 and Prolonged Information Fig. 10 are reported in Supplementary Tables 1 and a couple of.

Information have been assessed for normality and equality of variances utilizing Shapiro–Wilk and Brown–Forsythe assessments, respectively. Two teams of usually distributed knowledge have been in contrast utilizing two-tailed Pupil’s t-tests (paired if relevant). Two teams of nonparametric knowledge have been in contrast utilizing two-tailed Mann–Whitney U-tests for impartial datasets and utilizing Wilcoxon matched-pairs signed-ranks check for paired datasets. Variations between the technique of a number of impartial teams of information that met the assumptions of normality and equality of variances have been in contrast utilizing one-way ANOVA adopted by Holm–Šídák’s multiple-comparisons check. The place the assumptions of normality or of equality of variances have been violated, group means have been in contrast utilizing Kruskal–Wallis assessments adopted by Dunn’s multiple-comparisons check or by Welch’s ANOVA adopted by Dunnett’s T3 multiple-comparisons check, respectively. Reported P values have been corrected for a number of comparisons. Information proven in Fig. 2e have been analysed utilizing two-way repeated measures ANOVA with Geisser–Greenhouse correction. For a number of comparisons with parental controls, the very best of two P values was reported within the determine legend.

No pattern measurement calculations have been carried out earlier than experimentation. Pattern sizes have been chosen to match or exceed customary pattern sizes within the discipline. Pattern sizes in electrophysiological experiments correspond to the variety of cells, every of which was recorded in a special animal. Pattern sizes in behavioural experiments correspond to the variety of flies. The investigators weren’t blinded. Randomization was not relevant, as a result of flies have been grouped on the idea of genotype. In open-loop behavioural experiments (Fig. 5d–f) and all experiments involving two instructions of visible stimuli, stimulus instructions have been alternated randomly; the entire remaining visible stimuli have been introduced in a strict sequence to allow fast, intuitive interpretation (Figs. 1f and 5b). Two wild-type neurons have been misplaced after the third glutamate utility throughout patch-clamp recordings for Fig. 2e and have been excluded from the repeated-measures evaluation. Six cells have been misplaced throughout voltage-clamp experiments proven in Fig. 2f and Prolonged Information Fig. 4b as a consequence of pneumatic ejection. The present–voltage relationships of these cells don’t embrace all, however at the least six, knowledge factors per cell.

Reporting abstract

Additional info on analysis design is out there within the Nature Analysis Reporting Abstract linked to this paper.