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Surgir

Jul 18, 2023Jul 18, 2023

Nature volume 619, páginas 563–571 (2023)Cite este artigo

6541 Acessos

109 Altmétrico

Detalhes das métricas

Embora tenha havido progresso na identificação de sinais neurais relacionados a decisões rápidas e informadas1,2,3, menos se sabe sobre como o cérebro guia e finaliza decisões mais etologicamente relevantes, nas quais o próprio comportamento de um animal governa as opções experimentadas ao longo de minutos4,5, 6. Drosophila procura por muitos segundos a minutos locais de postura de ovos com alto valor relativo7,8 e possui neurônios, chamados oviDNs, cuja atividade preenche critérios de necessidade e suficiência para iniciar o programa motor de deposição de ovos9. Aqui mostramos que os oviDNs expressam um sinal de cálcio que (1) diminui quando um óvulo é preparado internamente (ovulação), (2) sobe e desce ao longo de segundos a minutos - de uma maneira influenciada pelo valor relativo dos substratos - como uma mosca. determina se deve pôr um ovo e (3) atinge um nível de pico consistente pouco antes do abdômen dobrar para a deposição do ovo. Este sinal é aparente nos corpos celulares dos oviDNs no cérebro e provavelmente reflete um processo de subida ao limiar comportamentalmente relevante no cordão nervoso ventral, onde os terminais sinápticos dos oviDNs estão localizados e onde sua saída pode influenciar o comportamento. Fornecemos evidências perturbacionais de que o programa motor de deposição de ovos é iniciado quando esse processo atinge um limite e que a variação subliminar nesse processo regula o tempo gasto considerando as opções e, em última análise, a escolha tomada. Finalmente, identificamos um pequeno circuito recorrente que alimenta os oviDNs e mostramos que a atividade em cada um dos seus tipos de células constituintes é necessária para a postura de um ovo. Esses resultados argumentam que um processo de subida ao limite regula uma decisão de valor relativo e individualizada e fornece uma visão inicial sobre o mecanismo de circuito subjacente para a construção desse processo.

A seleção do local de postura dos ovos é crítica para a sobrevivência da progênie de uma mosca10. Como tal, a Drosophila procura um substrato de alta qualidade durante muitos segundos a minutos antes de depositar cada ovo individual7,8. As preferências de postura de ovos para muitos substratos diferentes foram documentadas, mas não se sabe como os sinais neurais relacionados à decisão evoluem em tempo real para orientar o processo de seleção do local e para gerar essas preferências.

Gravamos vídeos de Drosophila grávida em uma pequena câmara com piso de substrato macio e caracterizamos uma sequência comportamental para postura de ovos (ver Tabelas Suplementares 1 e 2 para genótipos e condições em todos os experimentos). A sequência de seis passos começa com a mosca parada e realizando um alongamento do abdômen (etapa 1) seguido de um abdome (etapa 2) (Fig. 1a). A mosca então aumenta sua velocidade locomotora durante um período de busca (etapa 3) e, finalmente, realiza uma flexão de abdômen para deposição de ovos (etapa 4), deposita um ovo (etapa 5) e realiza uma segunda flexão de abdômen (etapa 6), provavelmente para limpeza do ovipositor.

a, Sequência comportamental de postura de ovos. b, ovo expressando GCaMP3 no corpo. As etapas correspondem a a. As inserções mostram close-ups, com pixels super/subsaturados em vermelho/azul; os painéis principais mostram pixels super/subsaturados em branco/preto. c, Progressão comportamental. As linhas conectam sequências únicas de postura de ovos. d, Esquema da roda. e, OviDNb único traçado a partir de imagens de microscopia óptica. A seta azul indica soma no cérebro, a seta verde indica saídas no gânglio abdominal. f, somas oviDN no lado direito do cérebro marcados por oviDN-SS1. g, oviDN ∆F/F e comportamento durante a postura de dois ovos pela mesma mosca. ∆F/F é suavizado com um filtro de vagão de 2 s. As imagens são projeções z de fatias de imagem selecionadas, com rótulos referentes a oviDNa e oviDNb (oviDNa é parcialmente obscurecido por oviDNb). h, OviDNb médio populacional ∆F/F alinhado ao final da curva abdominal para postura de ovos. O sombreamento cinza claro representa ±sem por toda parte; 43 traços de imagem de 41 eventos de postura de ovos associados a nove células em oito moscas. O número de traços excede o número de eventos de postura de ovos porque, para dois ovos, visualizamos oviDNb em ambos os lados do cérebro. Eventos comportamentais mostrados abaixo. i, Esquema da curvatura do abdômen. θ denota 'ângulo do corpo' e comprimento é a distância pescoço-ovipositor. j – l, média do oviDN ∆F/F e comportamento alinhado aos eventos em h: 'início da ovulação' (j), 'início da busca' (k) e conclusão da curvatura do abdômen (l). 'Comprimento normalizado' é o comprimento dado em i dividido por sua mediana (Métodos). Setas mais curtas e mais grossas indicam quando a curvatura do abdômen para deposição dos ovos está completa. Uma curva subsequente (mais forte) é, presumivelmente, para limpar o ovipositor. m, oviDN ∆F/F durante eventos individuais de postura de ovos, suavizados com um filtro de vagão de 5 s. Linha preta, quer dizer. n, OviDN médio ∆F/F durante a postura de ovos para todas as sete moscas que colocaram três ou mais ovos, suavizados com um filtro de vagão de 5 s. Uma única mosca GCaMP7b é mostrada em cinza. NP, Projeto Nippon; Avenida, média; 2-p, dois fótons; Éfis, eletrofisiologia; Máx., máximo.

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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