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Jul 18, 2023

Nature 619권, 563~571페이지(2023)이 기사 인용

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빠르고 단서가 있는 결정1,2,3과 관련된 신경 신호 식별에 진전이 있었지만, 동물 자신의 행동이 몇 분 동안 경험한 옵션을 지배하는 동물행동학적으로 더 관련된 결정을 뇌가 어떻게 안내하고 종료하는지에 대해서는 알려진 바가 적습니다4,5, 6. 초파리는 상대 가치가 높은 산란 장소를 몇 초에서 몇 분 동안 검색하며 oviDN이라는 뉴런을 가지고 있습니다. 이 뉴런의 활동은 알 침착 모터 프로그램을 시작하기 위한 필요성과 충분성 기준을 충족합니다9. 여기에서 우리는 oviDN이 (1) 난자가 내부적으로 준비(배란)될 때 낮아지고, (2) 기질의 상대적 가치에 영향을 받는 방식으로 몇 초에서 몇 분에 걸쳐 위아래로 표류하는 칼슘 신호를 파리처럼 표현한다는 것을 보여줍니다. 알을 낳을지 여부를 결정하고 (3) 알을 낳기 위해 복부를 구부리기 직전에 일관된 최고 수준에 도달합니다. 이 신호는 뇌에 있는 oviDN의 세포체에서 분명하게 나타나며 아마도 oviDN의 시냅스 말단이 위치하고 그 출력이 행동에 영향을 줄 수 있는 복부 신경 코드의 행동 관련 역치 상승 과정을 반영할 것입니다. 우리는 이 프로세스가 임계값에 도달하면 계란 증착 모터 프로그램이 시작되고 이 프로세스의 임계값 이하의 변화가 옵션을 고려하는 데 소요되는 시간과 궁극적으로 선택을 하는 시간을 조절한다는 교란적인 증거를 제공합니다. 마지막으로 우리는 oviDN에 공급되는 작은 순환 회로를 식별하고 각 구성 세포 유형의 활동이 알을 낳는 데 필요하다는 것을 보여줍니다. 이러한 결과는 임계값 상승 프로세스가 상대 가치, 자기 주도적 결정을 규제하고 이 프로세스를 구축하기 위한 기본 회로 메커니즘에 대한 초기 통찰력을 제공한다고 주장합니다.

알을 낳는 장소 선택은 파리 자손의 생존에 매우 중요합니다10. 따라서 Drosophila는 각 개별 알을 입금하기 전에 몇 초에서 몇 분 동안 고품질 기질을 검색합니다. 다양한 기질에 대한 알을 낳는 선호도가 문서화되어 있지만 결정 관련 신경 신호가 실시간으로 진화하여 사이트 선택 프로세스를 안내하고 이러한 선호도를 생성하는 방법은 알려져 있지 않습니다.

우리는 부드러운 기판 바닥이 있는 작은 챔버에서 임신 초파리의 비디오를 촬영하고 알을 낳는 행동 순서를 특성화했습니다(모든 실험의 유전자형 및 조건에 대해서는 보충 표 1 및 2 참조). 6단계 시퀀스는 파리가 가만히 서서 복부 신장(1단계)을 수행한 후 크런치(2단계)를 수행하는 것으로 시작됩니다(그림 1a). 그런 다음 파리는 검색 기간(3단계) 동안 이동 속도를 증가시키고, 마지막으로 알을 낳기 위해 복부 굽힘을 수행하고(4단계), 알을 낳고(5단계) 두 번째 복부 굽힘을 수행합니다(6단계). 산란관 청소용.

a, 알을 낳는 행동 순서. b, 체내에서 GCaMP3를 발현하는 Egg. 단계는 다음과 같습니다. 삽입은 빨간색/파란색의 과/과포화 픽셀이 있는 클로즈업을 보여줍니다. 메인 패널에는 흰색/검은색으로 과/과포화 픽셀이 표시됩니다. c, 행동 진행. 선은 단일 알을 낳는 순서를 연결합니다. d, 바퀴의 개략도. e, 광학 현미경 이미지에서 추적된 단일 oviDNb. 파란색 화살표는 뇌의 소마를 나타내고, 녹색 화살표는 복부 신경절의 출력을 나타냅니다. f, oviDN-SS1로 라벨링된 뇌 오른쪽의 oviDN 소마. g, oviDN ΔF/F 및 동일한 파리가 두 개의 알을 낳는 동안의 행동. ΔF/F는 2초 박스카 필터를 사용하여 평활화됩니다. 이미지는 oviDNa 및 oviDNb를 참조하는 레이블이 있는 선택된 이미징 슬라이스의 z 투영입니다(oviDNa는 oviDNb에 의해 부분적으로 가려짐). h, 인구 평균 oviDNb ΔF/F는 산란을 위해 복부 굴곡 끝 부분에 정렬됩니다. 밝은 회색 음영은 전체적으로 ±sem을 나타냅니다. 파리 8마리의 9개 세포와 관련된 41개의 산란 사건에서 나온 43개의 영상 흔적. 두 개의 알에 대해 뇌 양쪽에서 oviDNb를 이미지화했기 때문에 흔적의 수가 알을 낳는 사건의 수를 초과합니다. 아래에 표시된 동작 이벤트. i, 복부 굴곡의 도식. θ는 '신체 각도'를 나타내고 길이는 목-산란관 거리입니다. j–l, 평균 oviDN ΔF/F 및 h의 이벤트에 맞춰진 행동: '배란 시작'(j), '검색 시작'(k) 및 복부 굴곡 완료(l). '정규화된 길이'는 i에 주어진 길이를 중앙값(방법)으로 나눈 값입니다. 더 짧고 두꺼운 화살표는 난자 침착을 위한 복부 굽힘이 완료되었음을 나타냅니다. 이후의 (더 강한) 굽힘은 아마도 산란관을 청소하기 위한 것입니다. m, oviDN ΔF/F는 개별 산란 이벤트 중이며 5초 박스카 필터로 부드럽게 처리됩니다. 검은 선, 뜻. n, 3개 이상의 알을 낳은 파리 7마리 모두에 대해 알을 낳는 동안 평균 oviDN ΔF/F를 5초 박스카 필터로 부드럽게 처리했습니다. 단일 GCaMP7b 파리가 회색으로 표시됩니다. NP, 일본 프로젝트; 평균, 평균; 2-p, 2광자; 에피스(Ephys), 전기생리학; 최대, 최대.

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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