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Nouvelles

Surgir

Jul 09, 2023Jul 09, 2023

Nature volume 619, pages 563-571 (2023)Citer cet article

5643 Accès

113 Altmétrique

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Alors que des progrès ont été réalisés dans l'identification des signaux neuronaux liés à des décisions rapides et indiquées1,2,3, on en sait moins sur la façon dont le cerveau guide et met fin à des décisions plus pertinentes sur le plan éthologique dans lesquelles le comportement d'un animal régit les options expérimentées au fil des minutes4,5, 6. La drosophile recherche pendant plusieurs secondes, voire quelques minutes, des sites de ponte ayant une valeur relative élevée7,8 et possède des neurones, appelés oviDN, dont l'activité remplit les critères de nécessité et de suffisance pour lancer le programme moteur de dépôt des œufs9. Nous montrons ici que les oviDN expriment un signal calcique qui (1) diminue lorsqu'un œuf est préparé en interne (ovulé), (2) dérive de haut en bas en quelques secondes, voire minutes, d'une manière influencée par la valeur relative des substrats, comme une mouche. détermine s'il faut pondre un œuf et (3) atteint un niveau maximal constant juste avant la courbure de l'abdomen pour la déposition des œufs. Ce signal est apparent dans les corps cellulaires des oviDN dans le cerveau et reflète probablement un processus d'élévation du seuil comportemental dans la moelle nerveuse ventrale, où se trouvent les terminaisons synaptiques des oviDN et où leur production peut influencer le comportement. Nous fournissons des preuves perturbatrices que le programme moteur de dépôt des œufs est lancé une fois que ce processus atteint un seuil et que la variation infra-seuil de ce processus régule le temps passé à considérer les options et, finalement, le choix effectué. Enfin, nous identifions un petit circuit récurrent qui alimente les oviDN et montrons qu'une activité dans chacun de ses types cellulaires constitutifs est nécessaire à la ponte d'un œuf. Ces résultats soutiennent qu'un processus d'élévation du seuil régule une décision de valeur relative à votre rythme et fournissent un premier aperçu du mécanisme de circuit sous-jacent à la construction de ce processus.

La sélection du site de ponte est essentielle à la survie de la progéniture d'une mouche10. En tant que telle, la drosophile recherche un substrat de haute qualité pendant plusieurs secondes, voire quelques minutes, avant de déposer chaque œuf individuel7,8. Les préférences en matière de ponte pour de nombreux substrats différents ont été documentées10, mais la manière dont les signaux neuronaux liés à la décision évoluent en temps réel pour guider le processus de sélection du site et générer ces préférences est inconnue.

Nous avons pris des vidéos de drosophiles gravides dans une petite chambre avec un substrat mou et caractérisé une séquence comportementale pour la ponte (voir les tableaux supplémentaires 1 et 2 pour les génotypes et les conditions de toutes les expériences). La séquence en six étapes commence avec la mouche immobile et effectuant une élongation de l'abdomen (étape 1) suivie d'un scrunch (étape 2) (Fig. 1a). La mouche augmente ensuite sa vitesse locomotrice pendant une période de recherche (étape 3), et enfin elle effectue une courbure de l'abdomen pour la ponte des œufs (étape 4), dépose un œuf (étape 5) et effectue une deuxième courbure de l'abdomen (étape 6), probablement pour nettoyer l'ovipositeur.

a, Séquence comportementale de la ponte. b, Oeuf exprimant GCaMP3 dans le corps. Les étapes correspondent à a. Les encarts montrent des gros plans, avec des pixels sur/sous-saturés en rouge/bleu ; les panneaux principaux affichent des pixels sur/sous-saturés en blanc/noir. c, progression comportementale. Les lignes relient des séquences de ponte uniques. d, Schéma de la roue. e, oviDNb unique tracé à partir d’images de microscopie optique. La flèche bleue indique le soma dans le cerveau, la flèche verte indique les sorties dans le ganglion abdominal. f, somas oviDN sur le côté droit du cerveau marqué par oviDN-SS1. g, oviDN ∆F/F et comportement lors de la ponte de deux œufs par la même mouche. ∆F/F est lissé avec un filtre wagon couvert de 2 s. Les images sont une projection en Z de tranches d’imagerie sélectionnées, avec des étiquettes faisant référence à oviDNa et oviDNb (oviDNa est partiellement masqué par oviDNb). h, oviDNb moyenné par la population ∆F/F aligné à l’extrémité de la courbure de l’abdomen pour la ponte. L'ombrage gris clair représente ±sem partout ; 43 traces d'imagerie provenant de 41 événements de ponte associés à neuf cellules dans huit mouches. Le nombre de traces dépasse le nombre d’événements de ponte car pour deux œufs, nous avons imagé l’oviDNb des deux côtés du cerveau. Événements comportementaux présentés ci-dessous. i, Schéma de la courbure de l'abdomen. θ désigne « l'angle du corps » et la longueur correspond à la distance cou-ovipositeur. j–l, oviDN moyen ∆F/F et comportement aligné sur les événements de h : « début de l'ovulation » (j), « début de la recherche » (k) et achèvement de la courbure de l'abdomen (l). La « longueur normalisée » est la longueur donnée en i divisée par sa médiane (Méthodes). Des flèches plus courtes et plus épaisses indiquent quand la courbure de l'abdomen pour la ponte des œufs est terminée. Un virage ultérieur (plus fort) est probablement destiné au nettoyage de l'ovipositeur. m, oviDN ∆F/F lors d'événements de ponte individuels, lissés avec un filtre wagon couvert de 5 s. Ligne noire, méchante. n, oviDN moyen ∆F/F pendant la ponte pour les sept mouches ayant pondu trois œufs ou plus, lissé avec un filtre wagon couvert de 5 s. Une seule mouche GCaMP7b est représentée en gris. NP, Projet Nippon ; Ave., moyenne ; 2-p, deux photons ; Ephys, électrophysiologie ; Max., maximum.

Kir2.1* flies) could still lay eggs, albeit at lower mean levels compared with genetic-background-matched controls (Fig. 5c and Methods). Whole-cell, patch-clamp recordings showed that Kir2.1*-expressing oviDNs (or oviDN-like neurons) were hyperpolarized by around 14 mV, on average, compared with Kir2.1*Mut-expressing (control) cells (Fig. 5d). This is a moderate hyperpolarization that still permitted most Kir2.1*-expressing neurons to fire spikes with sufficient current injection (Extended Data Fig. 10d). This fact could explain why many oviDN>Kir2.1* flies could lay eggs./p>Kir2.1*Mut (e) and oviDN-GAL4>Kir2.1* (f) flies. Each row represents a single egg-laying event in a 0 versus 200 mM sucrose chamber, aligned to egg deposition, with the fly’s speed indicated by intensity of black shading. Rows ordered based on the search duration; 1,377 eggs from 40 flies (45 flies tested, of which five did not lay eggs) and 346 eggs from 17 flies (40 flies tested, of which 23 did not lay eggs), respectively. g, Median duration of search for individual flies from e,f that laid five or more eggs. Mean ± s.e.m., P = 9.6 × 10–7. h, Fraction of time spent walking during non-egg-laying periods for flies shown in g. Non-egg-laying periods were defined as periods of over 10 min from egg deposition. i, Fraction of eggs on the lower-sucrose option with 95% confidence interval. Each dot represents one fly. Individual flies laid an average of 38, 38, 32, 16, six and seven eggs each. If the plot is reworked by examining only flies that laid at least five eggs, P = 1.9 × 10–6 (rather than 6.3 × 10–4) for the middle set of bars and is not significant (NS) for the others. g–i, P values calculated using two-sided Wilcoxon rank-sum test. c–i, Tubulin>GAL80ts was present in all flies, to limit the time window in which Kir2.1* or Kir2.1*Mut transgenes were expressed (Methods). The 18 °C control was not shifted to 31 °C before the assay and thus expression of Kir2.1* or Kir2.1*Mut was not induced. All egg-laying experiments were conducted at 24 °C./p>Kir2.1* and oviDN>Kir2.1*Mut flies in two-substrate, free-behaviour chambers. We observed a two- to threefold increase in the length of the search period in oviDN>Kir2.1* compared with oviDN>Kir2.1*Mut flies when comparing the full distribution of traces from all flies (P < 0.001; Fig. 5e,f and Methods), or when quantifying median search duration per fly (comparing flies that laid sufficient eggs for analysis—that is, at least five eggs; Fig. 5g). The increase in search duration could not be attributed to a general increase in the fraction of time spent walking (Fig. 5h), nor to a broad defect in egg-laying-related motor functions (Extended Data Fig. 10e,f). Remarkably, just as we imagined, the increase in search duration was accompanied by a higher fraction of eggs laid on the substrate of higher relative value (Fig. 5i), probably because oviDN>Kir2.1* flies have more time to encounter the higher-relative-value option before threshold is reached./p> 5 min. away from egg deposition, i.e., ‘non-egg-laying periods’. b, Example trace of wheel position and oviDN ∆F/F during a non-egg-laying period (smoothed with a 2 s boxcar filter). This cell had a standard deviation in ∆F/F of 0.15. c, Mean cross-correlation of oviDN ∆F/F versus varied behavioral measures during non-egg-laying periods. Light grey shading is ± s.e.m. for all panels in this figure. For sucrose concentration correlations, only 0 vs. 500 mM sucrose wheels were analyzed (excluding 0 mM only wheels, for example), leaving 53/104 flies for analysis. d, Same as panel c, but including time periods near egg deposition (~372 additional minutes—i.e., ~4% additional sample points—are included compared to panel c). e, Mean oviDN ∆F/F and behavior during peaks in ∆F/F that occurred in non-egg-laying periods. We smoothed the ∆F/F signal with a 5 s boxcar filter and extracted peaks in the ∆F/F trace that exceeded 0.35 for > 1 s. We aligned these traces to the moment the ∆F/F signal crossed 0.35 in the 10 s before the peak. f, Change in mean body angle, replotted from Fig. 2h. Arrow indicates first bin with an abdomen angle change greater than 2.5° (indicated by dotted line). g, Same as panel f but with coarser binning. h, i, Same as panel f but with finer binning. j-n, Same as panel f but bins are shifted progressively by 0.02 leftward. In panels f to n, the first and last bin always include all the data points below and above that bin, respectively. The curve in panel l appears less step-like than the others; however, it is expected that as one progressively shifts the center point of the bins, one will find a position where the central bin straddles the putative threshold, yielding an intermediate y value for that bin. The fact that panels k and m appear more step like supports this explanation for panel l. o, Example traces of oviDN ∆F/F during prolonged, gentle CsChrimson stimulation (protocol described in Methods), smoothed with a 2.5 s boxcar filter. Traces are clipped once they reach a ∆F/F of 0.275. We used 0.275 as the threshold because it is slightly higher than the center of the 4th bin in Fig. 2g, h (i.e., a conservative lower-bound estimate of the threshold). We use a conservative estimate for this analysis to capture as many relevant traces as possible. Note that for a variety of reasons, CsChrimson expressing flies may have a different threshold in terms of ∆F/F than flies not expressing CsChrimson (Methods). OviDN ∆F/F traces occasionally rise to threshold with this protocol. p, OviDN ∆F/F smoothed with a 2.5 s boxcar filter for all 27 stimulations (out of 127 total) that brought ∆F/F to threshold during the stimulation interval (the other 100 stimulations that did not bring ∆F/F to the threshold are not shown). The beginning of each trace is the beginning of stimulation. Colored lines are traces from panel o. A similar analysis in the inter-stimulation-interval (starting 10 s after the CsChrimson stimulation ended) only identifies 2 threshold crossing events indicating that the observed threshold crossing during stimulation was predominantly caused by the stimulation (data not shown). A similar analysis using data with the strongest 5 s stimulation intensity in Fig. 2f identifies 46 (out of 88 total) threshold crossing events indicating that is harder to achieve threshold crossing with the gentle prolonged stimulation despite the longer interval (data not shown). q, r, Change in mean body length and body angle for data shown in panel p, indicating that flies, on average, bend their abdomen proximal to the time of threshold crossing. s, Remaining ∆F/F until threshold is reached (y-axis) as a function of remaining time until threshold is reached (x-axis). The traces in panel p are sampled at 100 ms intervals to populate bin counts of the histogram. The negative correlation indicates that CsChrimson stimulation gradually brings the ∆F/F to threshold, rather than by inducing a spontaneous event, independent of the current ∆F/F, that brings ∆F/F to threshold./p> 2 mm away from the boundary between two substrates (y axis), as a function of time from the substrate crossing (x axis). For a 2.5 mm fly, not being in the 2 mm region surrounding the boundary corresponds to the front or back of the fly being 0.75 mm away from the midpoint of the 1 mm plastic barrier between substrates. These traces highlight that it takes flies ~10–20 s, on average, to completely cross the midline which is important to keep in mind when interpreting neural signals aligned to substrate crossing events. b, Mean neck to proboscis length during substrate transitions. Light grey shading is ± s.e.m. for all panels in this figure. c, Mean locomotor speed during substrate transitions. d, Mean body length during substrate transitions. e, Mean body angle during substrate transitions. f, Mean body length, body angle, and oviDN ∆F/F during the subset of substrate transitions where there was a small change in body length. The mean body length in the 4 s after and before a substrate transition were subtracted. If the absolute value of this difference was less than 0.01, then the change was considered small. g, Same as panel f, except selecting for substrate transitions where the difference was greater than 0.01. h, Same as panel f, except selecting for substrate transitions where the difference was less than −0.01. The sum of the number of traces in panels f-h is less than panel a because during some substrate transitions the body length and/or angle was not possible to accurately calculate using DeepLabCut (Methods). i–k, Same as panels f-h, except comparing body angle and using a threshold of 0.5°. Proboscis length and fly speed (panels b-c) do not consistently change during substrate transitions and therefore do not explain the changes in oviDN ∆F/F. Body length and body angle do change, on average, during substrate transitions (panels d-e). However, these changes cannot fully explain the changes in oviDN ∆F/F (panels f-k). That is, regardless of the change in body length or body angle, the oviDN ∆F/F consistently changes with sucrose concentration (albeit with some modulations related to body length and angle)./p>Kir2.1* flies is indicative of the longer search duration in these flies. However, other aspects like the pause to lay an egg and post-egg-laying speed remain similar in oviDN>Kir2.1*Mut and oviDN>Kir2.1* flies. 1377 eggs from 40 flies (45 flies tested and 5 laid no eggs), 346 eggs from 17 flies (40 flies tested and 23 laid no eggs) for oviDN>Kir2.1*Mut and oviDN>Kir2.1*, respectively. f, Normalized inter-egg interval histograms. 1340 intervals from 40 oviDN>Kir2.1*Mut flies (45 flies tested and 5 laid < 2 eggs and thus did not have at least one interval). 333 intervals from 15 oviDN>Kir2.1* flies (40 flies tested and 25 flies laid < 2 eggs and thus did not have at least one interval). Note that the similar inter-egg interval distribution for oviDN>Kir2.1* and control flies does not mean that oviDN>Kir2.1* flies searched for the same amount of time for an egg-laying substrate as controls; rather, oviDN>Kir2.1* flies searched longer than controls (Fig. 5g). What is going on, remarkably, is that oviDN>Kir2.1* flies perform their next ovulation sooner after laying an egg than controls, such that despite searching longer before laying an egg, these flies ended up expressing nearly identical inter-ovulation and inter-egg intervals as control flies. The inter-ovulation interval (as estimated with locomotor speed) was not statistically different in oviDN>Kir2.1* and control flies (P = 0.36) (data not shown). P-values were calculated using two-sided Wilcoxon rank sum test./p>