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Presentarsi

Jul 18, 2023Jul 18, 2023

Natura volume 619, pagine 563–571 (2023) Citare questo articolo

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Mentre sono stati compiuti progressi nell'identificazione dei segnali neurali legati a decisioni rapide e guidate1,2,3, si sa meno su come il cervello guidi e interrompa decisioni più rilevanti dal punto di vista etologico in cui il comportamento di un animale governa le opzioni sperimentate nel corso di minuti4,5, 6. La drosophila cerca per molti secondi o minuti siti di deposizione delle uova con un elevato valore relativo7,8 e possiede neuroni, chiamati oviDN, la cui attività soddisfa i criteri di necessità e sufficienza per avviare il programma motorio di deposizione delle uova9. Qui mostriamo che gli oviDN esprimono un segnale di calcio che (1) diminuisce quando un uovo viene preparato internamente (ovulato), (2) va su e giù per secondi o minuti, in modo influenzato dal valore relativo dei substrati, come una mosca determina se deporre un uovo e (3) raggiunge un livello di picco costante appena prima che l'addome si pieghi per la deposizione delle uova. Questo segnale è evidente nei corpi cellulari degli oviDN nel cervello e probabilmente riflette un processo di aumento della soglia comportamentale rilevante nel cordone nervoso ventrale, dove si trovano i terminali sinaptici degli oviDN e dove la loro uscita può influenzare il comportamento. Forniamo prove perturbative che il programma motorio di deposizione delle uova viene avviato una volta che questo processo raggiunge una soglia e che la variazione sottosoglia in questo processo regola il tempo impiegato a considerare le opzioni e, in definitiva, la scelta presa. Infine, identifichiamo un piccolo circuito ricorrente che alimenta gli oviDN e mostriamo che l'attività in ciascuno dei suoi tipi di cellule costituenti è necessaria per deporre un uovo. Questi risultati sostengono che un processo di raggiungimento della soglia regola una decisione di valore relativo e autogestita e forniscono una visione iniziale del meccanismo del circuito sottostante per la costruzione di questo processo.

La selezione del sito di deposizione delle uova è fondamentale per la sopravvivenza della progenie di una mosca10. Pertanto, la Drosophila cerca un substrato di alta qualità per molti secondi o minuti prima di depositare ogni singolo uovo7,8. Sono state documentate le preferenze di deposizione delle uova per molti substrati diversi10, ma non è noto il modo in cui i segnali neurali legati alle decisioni si evolvono in tempo reale per guidare il processo di selezione del sito e per generare queste preferenze.

Abbiamo filmato la Drosophila gravida in una piccola camera con un pavimento di substrato morbido e caratterizzato una sequenza comportamentale per la deposizione delle uova (vedere Tabelle Supplementari 1 e 2 per i genotipi e le condizioni in tutti gli esperimenti). La sequenza in sei fasi inizia con la mosca ferma ed eseguendo un allungamento dell'addome (fase 1) seguito da uno scricchiolio (fase 2) (Fig. 1a). La mosca quindi aumenta la sua velocità locomotrice durante un periodo di ricerca (fase 3), e infine esegue una piegatura dell'addome per la deposizione delle uova (fase 4), deposita un uovo (fase 5) ed esegue una seconda piegatura dell'addome (fase 6), probabilmente per pulire l'ovopositore.

a, Sequenza comportamentale della deposizione delle uova. b, Uovo che esprime GCaMP3 nel corpo. I passaggi corrispondono a a. Gli inserti mostrano primi piani, con pixel sovra/sottosaturi in rosso/blu; i pannelli principali mostrano pixel sovra/sottosaturi in bianco/nero. c, Progressione comportamentale. Le linee collegano singole sequenze di deposizione delle uova. d, schema della ruota. e, Singolo oviDNb tracciato da immagini al microscopio ottico. La freccia blu indica il soma nel cervello, la freccia verde indica le uscite nel ganglio addominale. f, somi oviDN sul lato destro del cervello etichettati da oviDN-SS1. g, oviDN ∆F/F e comportamento durante la deposizione di due uova da parte della stessa mosca. ∆F/F viene livellato con un filtro boxcar da 2 s. Le immagini sono proiezioni z di sezioni di imaging selezionate, con etichette che si riferiscono a oviDNa e oviDNb (oviDNa è parzialmente oscurato da oviDNb). h, oviDNb medio della popolazione ∆F/F allineato all'estremità della curvatura dell'addome per la deposizione delle uova. L'ombreggiatura grigio chiaro rappresenta ±sem ovunque; 43 tracce di imaging da 41 eventi di deposizione delle uova associati a nove cellule in otto mosche. Il numero di tracce supera il numero di eventi di deposizione delle uova perché per due uova abbiamo ripreso oviDNb su entrambi i lati del cervello. Eventi comportamentali mostrati di seguito. i, Schema della curvatura dell'addome. θ denota "angolo del corpo" e la lunghezza è la distanza collo-ovopositore. j–l, media oviDN ∆F/F e comportamento allineato agli eventi in h: "inizio dell'ovulazione" (j), "inizio della ricerca" (k) e completamento della curvatura dell'addome (l). La "lunghezza normalizzata" è la lunghezza indicata in i divisa per la sua mediana (metodi). Le frecce più corte e più spesse indicano quando la piegatura dell'addome per la deposizione delle uova è completa. Una successiva piegatura (più forte) serve presumibilmente per pulire l'ovopositore. m, oviDN ∆F/F durante i singoli eventi di deposizione delle uova, livellati con un filtro per vagone merci da 5 s. Linea nera, cattivo. n, media oviDN ∆F/F durante la deposizione delle uova per tutte e sette le mosche che hanno deposto tre o più uova, levigate con un filtro per vagone merci da 5 s. Una singola mosca GCaMP7b è mostrata in grigio. NP, Progetto Nippon; Ave., media; 2-p, due fotoni; Efisologia, elettrofisiologia; Massimo, massimo.

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>

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