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Jul 09, 2023

Nature volume 619、pages 563–571 (2023)この記事を引用

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迅速な手がかりに基づく意思決定に関連する神経信号の同定は進歩している1、2、3が、動物自身の行動が数分にわたって経験する選択肢を支配する、より動物行動学的に関連性の高い意思決定を脳がどのように導き、終了させるのかについてはあまり知られていない4、5。 6. ショウジョウバエは、相対価値の高い産卵部位を数秒から数分かけて探します 7,8 。また、oviDN と呼ばれるニューロンがあり、その活動は産卵運動プログラムを開始するための必要性および十分性の基準を満たしています 9。今回我々は、oviDNが、(1) 卵子が体内で準備される(排卵される)ときに低下する、(2) 基質の相対値の影響を受けて、数秒から数分間にわたって上下に変動するカルシウムシグナルをハエとして発現することを示す。卵を産むかどうかを決定し、(3) 産卵のために腹部が曲がる直前に一貫したピークレベルに達します。このシグナルは脳内の oviDN の細胞体で明らかであり、おそらく、oviDN のシナプス末端が位置し、その出力が行動に影響を与える腹側神経索における行動に関連した閾値上昇プロセスを反映していると考えられます。我々は、このプロセスが閾値に達すると卵子沈着運動プログラムが開始され、このプロセスにおける閾値以下の変動が選択肢を検討するのに費やされる時間を制御し、最終的には選択が行われることを示す摂動的な証拠を提供する。最後に、oviDN に栄養を与える小さな回帰回路を特定し、その構成細胞タイプのそれぞれの活動が産卵に必要であることを示します。これらの結果は、閾値上昇プロセスが相対値で自分のペースで行う決定を制御し、このプロセスを構築するための基礎となる回路メカニズムへの最初の洞察を提供することを主張します。

産卵場所の選択は、ハエの子孫の生存にとって重要です10。そのため、ショウジョウバエは、個々の卵を産む前に、数秒から数分かけて高品質の基質を探します7,8。多くの異なる基質に対する産卵の好みは文書化されている 10 が、意思決定に関連する神経信号がリアルタイムでどのように進化して場所の選択プロセスを導き、これらの好みを生成するかは不明です。

柔らかい基質床を備えた小さな部屋で妊娠中のショウジョウバエのビデオを撮影し、産卵の行動シーケンスを特徴付けました（すべての実験の遺伝子型と条件については補足表1および2を参照）。 6 ステップのシーケンスは、ハエが静止して腹部を伸ばす動作 (ステップ 1) に続いてスクランチ (ステップ 2) を行うことから始まります (図 1a)。その後、ハエは探索期間中に運動速度を上げ (ステップ 3)、最後に卵を産むために腹部を曲げ (ステップ 4)、卵を産み (ステップ 5)、2 回目の腹部を曲げる (ステップ 6) と考えられます。産卵器の掃除に。

a、産卵の行動順序。 b、体内でGCaMP3を発現している卵。手順は a に対応します。挿入図は、赤/青の過飽和/過飽和のピクセルを含むクローズアップを示しています。メインパネルには、白/黒で過飽和/過飽和のピクセルが表示されます。 c、行動の進行。線は単一の産卵シーケンスを接続します。 d、ホイールの概略図。 e、光学顕微鏡画像から追跡された単一のoviDNb。青い矢印は脳の細胞体を示し、緑色の矢印は腹部神経節の出力を示します。 f、oviDN-SS1で標識された脳の右側のoviDN細胞体。 g、oviDN ∆F/F および同じハエによる 2 つの卵の産卵中の行動。 ∆F/F は 2 秒のボックスカーフィルターで平滑化されます。画像は選択したイメージングスライスの Z 投影であり、oviDNA および oviDNb を参照するラベルが付いています (oviDNa は oviDNb によって部分的に隠されています)。 h、母集団平均 oviDNb ∆F/F は、産卵のための腹部の曲がりの端に位置合わせされています。明るい灰色の陰影は全体の±semを表します。 8 匹のハエの 9 個の細胞に関連する 41 回の産卵イベントからの 43 個の画像トレース。 2 個の卵について脳の両側の oviDNb を画像化したため、トレースの数は産卵イベントの数を超えています。以下に示す行動イベント。 i、腹部の曲がりの概略図。 θは「体の角度」を示し、長さは首と産卵器の距離を表します。 j–l、平均oviDN ∆F/F、およびhのイベントに合わせた行動：「排卵開始」（j）、「検索開始」（k）、および腹部屈曲の完了（l）。「正規化された長さ」は、i で指定された長さをその中央値で割ったものです (方法)。短くて太い矢印は、卵を産むための腹部の曲がりが完了する時期を示します。その後の（より強い）曲げは、おそらく産卵管を掃除するためです。 m、個々の産卵イベント中の oviDN ∆F/F、5 秒ボックスカーフィルターで平滑化。黒い線、意味あります。 n、3 個以上の卵を産んだ 7 匹のハエすべての産卵中の平均 oviDN ΔF/F、5 秒ボックスカーフィルターで平滑化。単一の GCaMP7b ハエが灰色で示されています。 NP、日本プロジェクト; 平均、平均。 2-p、2光子。エフィス、電気生理学。最大、最大。

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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