Natura volume 613, pagine 534–542 (2023) Citare questo articolo
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Per sopravvivere, gli animali devono convertire le informazioni sensoriali in comportamenti appropriati1,2. La visione è un senso comune per individuare stimoli etologicamente rilevanti e guidare le risposte motorie3,4,5. Il modo in cui i circuiti convertono la posizione dell'oggetto nelle coordinate retiniche nella direzione del movimento nelle coordinate del corpo rimane in gran parte sconosciuto. Qui mostriamo attraverso il comportamento, la fisiologia, l'anatomia e la connettomica in Drosophila che la trasformazione visuomotoria avviene mediante la conversione di mappe topografiche formate dai dendriti dei neuroni di proiezione visiva (VPN) che rilevano le caratteristiche6,7 in gradienti di peso sinaptici di output VPN sui neuroni del cervello centrale . Dimostriamo come questo motivo gradiente trasforma la posizione anteroposteriore di uno stimolo visivo incombente nella fuga direzionale della mosca. Nello specifico, scopriamo che due neuroni postsinaptici a un tipo VPN reattivo promuovono direzioni di decollo opposte. I gradienti di peso sinaptico opposti su questi neuroni provenienti da VPN incombenti in diverse regioni del campo visivo convertono le minacce incombenti localizzate in fughe orientate correttamente. Per un secondo tipo di VPN a risposta incombente, dimostriamo risposte graduate lungo l'asse dorsoventrale. Mostriamo che questo motivo del gradiente sinaptico si generalizza in tutti i 20 tipi di cellule VPN primarie e molto spesso si presenta senza la topografia degli assoni VPN. I gradienti sinaptici possono quindi essere un meccanismo generale per convogliare le caratteristiche spaziali delle informazioni sensoriali in output motori diretti.
Per prendere una palla, girarsi quando viene chiamato o prendere una tazza, il nostro cervello deve dirigere non solo cosa fare, ma anche dove farlo. Inerente a questo processo è una "trasformazione sensomotoria"2,8,9 in cui la posizione di un oggetto rilevata nello spazio sensoriale, come la posizione sulla retina, viene convertita in direzione del movimento in coordinate motorie, come la direzione di un arto o di un'articolazione cambiamenti di angolo. Esistono prove considerevoli che le regioni cerebrali organizzate topograficamente in un'ampia gamma di specie codificano la posizione e l'identità degli oggetti visivi10,11,12,13; tuttavia, il modo in cui i modelli di connettività neurale trasmettono tali informazioni alle reti premotorie a valle e il modo in cui i programmi di sviluppo specificano questa connettività rimangono poco conosciuti.
Nella Drosophila, le VPN che hanno dendriti nel lobo ottico e terminali assonici nel cervello centrale rilevano caratteristiche visive etologicamente rilevanti, come il movimento di piccoli oggetti o l'incombere di oggetti scuri6,7,14,15,16,17, e sono vicini a l'interfaccia sensomotoria. Diversi tipi di VPN avviano comportamenti guidati visivamente6,18,19,20,21 e alcuni tipi di VPN fanno sinapsi direttamente su un sottoinsieme di ≈500 neuroni discendenti premotori (DN) per emicefalo la cui attivazione guida azioni motorie distinte22,23,24. Esistono 20-30 diversi tipi di VPN, ciascuno con una popolazione di 20-200 neuroni per emicefalo (Fig. 1a), con piccoli campi recettivi (20-40°) che insieme coprono lo spazio visivo6,15,16. I dendriti VPN nel lobo ottico formano quindi una mappa topografica dello spazio visivo e la posizione dell'oggetto sulla retina del volo è teoricamente codificata mediante la quale vengono eccitati i neuroni VPN all'interno di un dato tipo. Tuttavia, non è chiaro se e come queste informazioni spaziali vengano trasmesse ai partner a valle perché gli assoni di tutte le VPN all'interno di un dato tipo terminano in glomeruli stretti e distinti all'interno del cervello centrale (Fig. 1a) con poco25 o nessun6,15 ,26,27 topografia osservabile a livello di microscopia ottica. Tuttavia, diversi tipi di cellule VPN sono stati associati a comportamenti specifici della direzione, tra cui indietreggiare e voltarsi, sfuggire a stimoli incombenti da direzioni diverse, evitare collisioni e, in volo, voltare le spalle a uno stimolo visivo6,28,29,30. Qui esaminiamo come le informazioni visive specifiche della direzione vengono trasformate nelle reti premotorie a valle esplorando l'interfaccia partner VPN-postsinaptico utilizzando la microscopia elettronica (EM), la microscopia ottica, la fisiologia e il comportamento.
50 postsynaptic neurons typically innervate each optic glomerulus. Inset: EM-based reconstructions (hemibrain connectome27) of 71 LC4 VPNs (blue), a single LC4 neuron (red) and LC4 postsynaptic partner, GF DN (black). VNC, ventral nerve cord; D, dorsal; L, lateral; glom., glomerulus. Scale bar, 20 μm. b, Confocal projections of GFP (green) expression in seven DNs innervating the LC4 glomerulus (red dashed line). Grey, brain neuropils. Images adapted from ref. 24, CC BY 4.0 (n = 4 brains for each DN). Scale bar, 50 μm. c, Synaptic connectivity from looming-sensitive VPN cell types onto seven DNs based on the hemibrain connectome. Arrow width is proportional to synapse number. Pie charts indicate proportion of a given DN's inputs from each looming-sensitive VPN cell type. d, Forward–backward postural shifts in response to DN photostimulation; quantified as Δ[T2 leg angle], the change in angle between the middle jumping legs and COM. e, Δ[T2 leg angle] 75 ms after the onset of 50-ms photostimulation. Points, individual flies; error bars, s.d.; one-way analysis of variance (ANOVA), Dunnett's test, ***P < 0.001, exact P values in Supplementary Table 1. f, Δ[T2 leg angle] time courses from machine-learning-tracked data; red shaded area, photostimulation period. g, Δ[T2 leg angle] for a subset of manually annotated flies. In f,g: lines, mean; shading, s.d. h, Takeoff direction is COM movement direction between onset of middle leg extension and takeoff. i, Polar histograms of optogenetically activated takeoff direction. Red line, circular mean; n, number of flies tested; \(\bar{R}\), mean vector length; P, Hodges–Ajne test for angular uniformity./p>90%), suggesting that natural threats may simultaneously activate multiple LC4-DNs to drive downstream escape motor circuits. DNp04- and DNp11-activated takeoffs were almost exclusively ‘long-mode’, in which the wings are raised before the takeoff jump, whereas GF activation produced ‘short-mode’ escapes without prior wing-raising as previously described36 (Extended Data Fig. 1g,h and Supplementary Table 1). Combination line activation drove primarily long-mode takeoff, but did also unexpectedly produce many short-mode takeoffs, which are thought to rely on GF activation. Taken together with the findings of our previous work37, this mixed result indicates either that the combination of DNp02, DNp04 and DNp06 inputs to the GFs, or that these DNs are not naturally co-activated with the strong intensity of optogenetic activation./p> 0.05). m, GF depolarization responses from localized activation of dorsal versus ventral LPLC2 and LC4 neurons expressing the P2X2 receptor. Left: representative GF responses (n = 5, one fly); individual (lighter-coloured lines) and averaged (darker lines) responses. Right: comparison of normalized average GF responses (resp.) to dorsal versus ventral VPN activation (two-tailed paired t-test; error bars, s.e.m., *P ≤ 0.05, ****P < 0.0001). Responses were averaged during the late response peak; see Extended Data Fig. 12c for quantification of the early peak. n, individual flies tested. A, anterior; P, posterior; D, dorsal; V, ventral; L, lateral; M, medial. All box plots show median and interquartile range./p> 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 for all figures where applicable. Statistical tests for Figs. 1e and 3e,h and Extended Data Figs. 1,2,4 and 12 are described in Supplementary Table 1. In all box plots (Fig. 6 and Extended Data Fig. 11), the solid line depicts the median; the upper and lower bounds of the box depict the third and first quantiles of the data spread, respectively. Whiskers indicate minimum and maximum values. All other statistical tests, number of replicates, statistical significance levels and other elements of statistical analysis are reported in the corresponding section of the Methods, along with the associated results and/or in the corresponding figure legends. No data were excluded from the analysis except as noted for the behaviour experiments (see the section in the Methods entitled Behavioural data analysis). All measurements were taken from distinct samples./p>Kir2.1. One trial per fly. b, Some flies also takeoff in response to looming, and those that do takeoff in a direction away from the stimulus (with some influence of the heading of the fly33). Shown are polar takeoff direction histograms with 12° bin width and mean resultant vector overlaid (red line). p, Hodges-Ajne test for angular uniformity. c, Takeoff direction results from the fly shifting its COM relative to the axes formed by a line connecting the ground contact points of its two middle jumping legs and a perpendicular bisector. Black points indicate COM at stimulus onset and red points indicate COM just prior to takeoff. d, The specific direction in which the COM moves in body coordinates depends on its starting location. Vector position is the COM position at stimulus onset. The vector itself indicates the shift of COM position from stimulus onset to just prior to takeoff. Black vectors are tracked data, gray vectors are interpolated. Black square is approximated point of convergence. e, Percent of flies (individual DN driver lines) that performed a takeoff in response to CsChrimson optogenetic activation in the FlyPEZ assay. Error bars, Wilson score interval; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs control (Empty, empty brain split-Gal4 control; DL – wild type control); normal approximation to binomial, two-sided Z-test, Bonferroni correction post hoc test. f, Same data as in (e) but with driver lines grouped by cell type. Error bars, SD. g, Histograms displaying the distribution of escape sequence durations between the wing raising and takeoff jump sub-behaviors (for LC4-DN driver lines expressing CsChrimson that can elicit escape upon activation). Escape trials are combined from split-Gal4 lines for each LC4-DN type. Short-mode escape duration (0 to 7 ms, gray shaded region) and long-mode escape duration (>7 ms), as previously established. h, Percentage of short-mode activated escapes. Error bars, Wilson score interval; ****p < 0.0001 versus GF; normal approximation to binomial, two-sided Z-test, Bonferroni correction post hoc test. Detailed description of statistical tests used and p-values for panels "e" and "h" is available in Supplementary Table 1./p> 0.1 for both DNs, Kuiper's Test). However, DN silencing altered the distribution of backward takeoffs direction in response to frontal looming (0°) for both DNp02 (p < 0.005, Kuiper's test) and DNp11 (p < 0.001, Kuiper's test) silencing compared to controls. Strikingly, many DNp02- and DNp11-silenced flies performed forward takeoffs in response to frontal looming stimulation, effectively jumping toward the threatening stimulus. c, To further understand why flies were inappropriately taking off forwards, we looked at how much DN-silenced flies moved their COM backwards in response to 0° looming. We visualized COM movement in body coordinates from different starting postures using the same flow fields in body-centric coordinates as in Extended Data Fig. 1d. Visual inspection indicated that COM movement fields for DN-silenced flies differed from controls in the amount of backwards movement and had more lateral movement. d, To quantify this motion, we measure the T2 angle (angle formed by T2 tarsal contact points and COM), which is >180° when the COM is in front of the T2 jumping legs and <180° when the COM is behind the T2 jumping legs. The mean T2 angle just before takeoff was significantly different for DNp02- and DNp11-silenced flies compared to controls (*p = 0.0468, ***p = 4.79e-04, One-Way ANOVA, Dunnett's test). Black points, individual flies; error bars, SD. e, Looking at time courses for T2 leg angle in response to 0° azimuth looming stimulus for the different DN-silenced lines (colors, shaded area, SD), with control data overlaid (grey), it is clear that the difference in the DN-silenced flies is that they do not shift backwards as much as controls. Since COM placement prior to takeoff determines whether the fly's jump will propel it forwards (T2 angle>180) or backwards (T2 angle<180), the impaired pre-takeoff T2 leg angle change in DNp02- and DNp11-silenced flies, which on average does not become <180° as in control flies, likely underlies altered takeoff performance leading to more forward-directed takeoffs. f, DNp02 and DNp11 silencing does not affect takeoff rates. Percentage of flies which performed a takeoff to a looming visual stimulus (azimuth = 90°, elevation = 45°) at four looming rates (l/v = 10, 20, 40 and 80 ms), or a looming visual stimulus (azimuth = 0° or 180°, elevation = 45°) at l/v = 40. L1/L2-silenced flies serve as "motion-blind" negative controls. Error bars, SEM; Wilson score interval; **p < 0.01, ***p < 0.001, ****p < 0.0001 versus Empty control; normal approximation to binomial, two-sided Z-test, Bonferroni correction post hoc test. Detailed description of statistical tests and p-values for panel "f" is available in Supplementary Table 1./p>50 synapses total. c, Representative examples of graded synaptic connectivity between four VPN cell types and their top 15 postsynaptic partners based on the total number of synapses. Each individual neuron within a VPN cell type is assigned a color based on just one plot (DNp11 for LC4, Giant Fiber for LPLC2 etc.), with the colors preserved in other graphs. Every plot indicates the number of synapses between individual neurons within one VPN cell type and a given postsynaptic partner (arranged by descending number of synapses). d, Single LC4 neurons (EM-based connectome reconstructions) with dendrites in anterior (bodyID 1907587934) or posterior (bodyID 1249932198) regions of the lobula are highlighted. The remaining LC4 neurons shown in grey. e, Differential synaptic connectivity between two LC4 neurons from (d) and their top 25 postsynaptic partners (measured by total number of synapses). 15 out of 25 postsynaptic neurons receive preferential or exclusive input from either anterior or posterior LC4. f–g, Differential synaptic connectivity of individual LPLC2 neurons with dendrites in dorsal (bodyID 1815826155) vs ventral (bodyID 1815809293) lobula. Similar to (d, e). P, posterior; M, medial; D, dorsal; L, lateral./p>0.05; *: P <0.05; **: P <0.01; ****: P <0.0001. Detailed description of statistical tests and p-values for panels is available in Supplementary Table 1./p>
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