AbstractThe neurexin superfamily, consisting of neurexins and Casprs, play important roles in the development, maintenance, function, and plasticity of neuronal circuits. Caspr/CNTNAP genes are linked to alterations in neuronal circuits and associated with neurodevelopmental and neurodegenerative conditions.

摘要神经毒素超家族由神经毒素和Casprs组成，在神经元回路的发育，维持，功能和可塑性中起着重要作用。Caspr/CNTNAP基因与神经元回路的改变有关，并与神经发育和神经退行性疾病有关。

Casprs are implicated in multiple neuronal signaling pathways, including dopamine; however, the molecular mechanisms by which Casprs differentially alter specific signaling pathways and downstream behaviors are unclear. We find that the C. elegans Caspr nlr-1 functions in neurons to control foraging behaviors, acting in distinct monoamine neurons to modulate locomotor activity in the presence or absence of food.

CASPR涉及多种神经元信号传导途径，包括多巴胺；然而，CASPR差异改变特定信号通路和下游行为的分子机制尚不清楚。我们发现秀丽隐杆线虫Caspr nlr-1在神经元中起控制觅食行为的作用，在不同的单胺神经元中起作用，在存在或不存在食物的情况下调节运动活动。

nlr-1 functions in dopamine neurons to reduce activity in the absence of food, similar to the role of dopamine, and regulates dopamine signaling through D2-like receptors. Furthermore, nlr-1 contributes to proper morphology and presynaptic structure of dopamine neurons, dopamine receptor expression and localization, and the behavioral response to dopamine.

nlr-1在多巴胺神经元中起作用，在没有食物的情况下降低活性，类似于多巴胺的作用，并通过D2样受体调节多巴胺信号传导。此外，nlr-1有助于多巴胺神经元的正确形态和突触前结构，多巴胺受体的表达和定位以及对多巴胺的行为反应。

We find that nlr-1 similarly regulates another dopamine-dependent behavior, the basal slowing response. Therefore, spatial manipulation of a broadly expressed neuronal gene is sufficient to alter neural circuits and behavior and uncovers important functions masked by global manipulation, highlighting the importance of genetic variation and mechanisms that impact spatial expression of genes to behavior..

我们发现nlr-1类似地调节另一种多巴胺依赖性行为，即基础减慢反应。因此，对广泛表达的神经元基因进行空间操作足以改变神经回路和行为，并揭示全球操作所掩盖的重要功能，突出了遗传变异的重要性以及影响基因空间表达与行为的机制。。

IntroductionStructural and functional properties of neuronal circuits underlie animal behavior, which can be altered through many modulatory mechanisms, including monoamine signaling pathways. The wiring of neuronal circuits requires proper coordination of synaptic adhesion molecules that facilitate the development and maturation of the connections between neurons at synapses and the maintenance of proper synapse function.

引言神经元回路的结构和功能特性是动物行为的基础，动物行为可以通过许多调节机制改变，包括单胺信号通路。神经元回路的布线需要适当协调突触粘附分子，以促进突触神经元之间连接的发育和成熟，并维持适当的突触功能。

The neurexin superfamily of genes, consisting of neurexins and contactin associated proteins (Casprs), are a major class of synaptic adhesion molecules required for the development and function of synapses. Humans have five Caspr genes (CNTNAP1, CNTNAP2, CNTNAP3, CNTNAP4, and CNTNAP5) and several of these genes have been linked to behavioral changes observed in individuals with autism and schizophrenia1,2,3,4,5,6,7.

神经毒素基因超家族由神经毒素和接触蛋白相关蛋白（Casprs）组成，是突触发育和功能所需的一类主要突触粘附分子。人类有五个Caspr基因（CNTNAP1，CNTNAP2，CNTNAP3，CNTNAP4和CNTNAP5），其中一些基因与自闭症和精神分裂症患者的行为改变有关1,2,3,4,5,6,7。

Additionally, Casprs have been linked to neurodegenerative diseases, with reduced Caspr2 expression found in the hippocampus of Alzheimer’s Disease patients8, decreased gene expression in the blood of patients with Parkinson’s Disease9 and increased cerebrospinal fluid and plasma expression of Caspr4 in patients with Parkinson’s Disease10.Mutations in CNTNAP gene orthologues lead to a variety of alterations in the nervous system in animal models.

此外，Casprs与神经退行性疾病有关，阿尔茨海默病患者海马Caspr2表达降低8，帕金森病患者血液中基因表达降低9，帕金森病患者脑脊液和血浆Caspr4表达增加10。CNTNAP基因直向同源物突变导致动物模型中神经系统的多种改变。

Mice lacking Cntnap2, Cntnap3, and Cntnap4 can have alterations in neural and synaptic development11,12,13,14,15,16, connectivity17, neuronal gene expression18, channel19 and receptor localization20, and neurophysiology21. Further, Cntnap2, Cntnap3, and Cntnap4 knockout mice have altered behaviors including social behaviors12,15,16,22,23,24, nest building16, hyperactivity15,24, repetitive behaviors12,15,16,24, and motor behaviors10,21.

缺乏Cntnap2，Cntnap3和Cntnap4的小鼠可能在神经和突触发育11,12,13,14,15,16，连接性17，神经元基因表达18，通道19和受体定位20以及神经生理学21方面发生改变。此外，Cntnap2，Cntnap3和Cntnap4基因敲除小鼠的行为发生了改变，包括社交行为12,15,16,22,23,24，筑巢16，多动15,24，重复行为12,15,16,24和运动行为10,21。

The conserved monoamine neurotransmitter dopamine, which plays.

保守的单胺类神经递质多巴胺，起作用。

nlr-1 functions in distinct monoamine neurons to modulate behavioral responses to food levelsWe were interested in the potential role of nlr-1 on the response to food and food deprivation in neurons and subsets of monoamine neurons. Complete loss of nlr-1 is embryonic/larval lethal39, so we obtained animals with a balanced heterozygous deletion in nlr-1.

nlr-1在不同的单胺神经元中起调节对食物水平的行为反应的作用我们对nlr-1在神经元和单胺神经元亚群中对食物和食物剥夺的反应中的潜在作用感兴趣。nlr-1的完全丧失是胚胎/幼虫致死39，因此我们获得了nlr-1中具有平衡杂合缺失的动物。

We also obtained animals with flippase recognition sites (frt) flanking regions of the endogenous nlr-1 gene, (coding and noncoding), allowing us to excise nlr-1 in a spatially restricted manner through cell-specific expression of the recombinase flippase (FLP) (Fig. 1A). Here we knocked out nlr-1 through expression of FLP in all neurons (rgef-1p::FLP), serotonin neurons (5-HT, tph-1p::FLP), dopamine neurons (DA, dat-1p::FLP), or octopamine neurons (OA, tbh-1p::FLP) (Supplementary Fig. 1B).

我们还获得了具有内源性nlr-1基因侧翼区域（编码和非编码）的翻转酶识别位点（frt）的动物，使我们能够通过重组酶翻转酶（FLP）的细胞特异性表达以空间受限的方式切除nlr-1）（图1A）。在这里，我们通过在所有神经元（rgef-1p：：FLP），5-羟色胺神经元（5-HT，tph-1p：：FLP），多巴胺神经元（DA，dat-1p：：FLP）或章鱼胺神经元（OA，tbh-1p：：FLP）（补充图1B）中表达FLP来敲除nlr-1。

This strain includes a mKate tag at the 3’ end of nlr-139, and we found that nlr-1 is expressed broadly in the nerve ring throughout development and into adulthood with some low-level expression in the pharynx (Supplementary Fig. 1C). Expressing FLP in all neurons eliminated expression of nlr-1 in the nerve ring (Supplementary Fig. 1D), and as previously shown, knocking out nlr-1 in neurons circumvents the lethality of homozygous deletions of nlr-1 globally39.Fig.

该菌株在nlr-139的3'端包含一个mKate标签，我们发现nlr-1在整个发育过程中和成年期在神经环中广泛表达，在咽部有一些低水平表达（补充图1C）。在所有神经元中表达FLP消除了nlr-1在神经环中的表达（补充图1D），并且如前所示，敲除神经元中的nlr-1规避了nlr-1全球纯合缺失的致死性39。

1: nlr-1 functions in distinct monoamine neurons to modulate behavioral responses to food levels.A Schematic of the human CNTNAP2 gene and the nlr-1 gene with the two alleles used in this study and cartoon of protein domains in CNTNAP2 and NLR-1 (DISC discoidin-like, FBG fibrinogen like, SP signal peptide, EGF epidermal growth factor like, LNS laminin/neurexin/sex hormone-binding globulin, TM transmembrane, FRT flippase recognition sites, mKate mKate fluorescent protein).

1： nlr-1在不同的单胺神经元中起作用，以调节对食物水平的行为反应。人CNTNAP2基因和nlr-1基因的示意图，以及本研究中使用的两个等位基因，以及CNTNAP2和nlr-1中蛋白质结构域的卡通（盘状蛋白样，FBG纤维蛋白原样，SP信号肽，EGF表皮生长因子样，LNS层粘连蛋白/神经毒素/性激素结合球蛋白，TM跨膜，FRT翻转酶识别位点，mKate-mKate荧光蛋白）。

A.

答：。

nlr-1 regulates the response to food deprivation through dopamine signalingWe wanted to further explore the potential role of nlr-1 in dopamine signaling suggested by the behavioral phenotype upon loss of nlr-1 in dopamine neurons and food deprivation (Fig. 2A). Dopamine is made from the conversion of tyrosine to L-DOPA by tyrosine hydroxylase (TH), and then conversion of L-DOPA to dopamine by DOPA-decarboxylase (DDC), with the rate-limiting TH enzyme encoded by the cat-2 gene in C.

nlr-1通过多巴胺信号调节对食物剥夺的反应我们想进一步探索nlr-1在多巴胺神经元和食物剥夺中nlr-1缺失时行为表型所提示的多巴胺信号传导中的潜在作用（图2A）。多巴胺是由酪氨酸羟化酶（TH）将酪氨酸转化为左旋多巴，然后通过多巴脱羧酶（DDC）将左旋多巴转化为多巴胺，其中限速TH酶由C中的cat-2基因编码。

elegans. We find that animals lacking dopamine due to a mutation in cat-2/TH have increased activity off food compared to controls, with no difference in activity on food (Fig. 2B). This behavioral phenotype is the same as observed in animals lacking nlr-1 in dopamine neurons. To determine whether nlr-1 mediates the response to food deprivation by directly impacting dopamine signaling, we generated animals lacking both cat-2 and nlr-1 in dopamine neurons.

线虫。我们发现，与对照组相比，由于cat-2/TH突变而缺乏多巴胺的动物在食物外的活性增加，而在食物上的活性没有差异（图2B）。这种行为表型与在多巴胺神经元中缺乏nlr-1的动物中观察到的相同。为了确定nlr-1是否通过直接影响多巴胺信号传导来介导对食物剥夺的反应，我们在多巴胺神经元中产生了缺乏cat-2和nlr-1的动物。

These animals had increased activity off food compared to controls, similar to animals lacking nlr-1 in dopamine neurons (Fig. 2C), which suggests that nlr-1 regulates dopamine signaling from dopamine neurons to regulate activity levels off food. Prior studies identified roles for nlr-1 in gap junction formation39.

与对照组相比，这些动物的食物外活动增加，类似于多巴胺神经元中缺乏nlr-1的动物（图2C），这表明nlr-1调节多巴胺神经元的多巴胺信号传导以调节食物外的活动水平。先前的研究确定了nlr-1在间隙连接形成中的作用39。

To test the possibility that nlr-1 may alter locomotion in response to food deprivation through gap junctions, we expressed a dominant negative form of UNC-940 in dopamine neurons using the dat-1 promoter. Expression of dominant negative UNC-9 reduced activity in the absence of food, in stark contrast to the phenotype observed with loss of nlr-1 in dopamine neurons (Supplementary Fig. 2G), indicating that nlr-1 likely does not modulate activity levels in the absence of food through gap junctions.Fig.

为了测试nlr-1可能通过间隙连接改变运动以响应食物剥夺的可能性，我们使用dat-1启动子在多巴胺神经元中表达了UNC-940的显性负性形式。显性负性UNC-9的表达在没有食物的情况下降低了活性，与多巴胺神经元中nlr-1缺失所观察到的表型形成鲜明对比（补充图2G），表明nlr-1可能不调节活性水平在没有食物的情况下通过间隙连接。图。

2: nlr-1 regulates the respon.

2:nlr-1调节反应。

nlr-1 organizes dopamine neuron and synaptic morphologySynaptic adhesion molecules can impact circuits through many mechanisms, including modification of neuron and synaptic structure and morphology. Casprs can localize and function in axons11,19, so we asked whether loss of nlr-1 impacts dopamine neuron and presynaptic morphology.

nlr-1组织多巴胺神经元和突触形态突触粘附分子可以通过许多机制影响电路，包括神经元和突触结构和形态的修饰。Casprs可以在轴突中定位和发挥功能11,19，因此我们询问nlr-1的缺失是否会影响多巴胺神经元和突触前形态。

We used fluorescent labeling of all dopamine neurons using expression of GFP under the dat-1 promoter (dat-1p::GFP) to visualize dopamine neuron morphology in controls and animals lacking nlr-1 in all neurons or lacking in just dopamine neurons. We noticed that the CEP neurons and axons appeared to have defects in nerve ring fasciculation in animals lacking nlr-1 in all neurons and in animals lacking nlr-1 in dopamine neurons (Fig. 2D, E and Supplementary Fig. 2D, E).

我们使用荧光标记所有多巴胺神经元，使用dat-1启动子（dat-1p：：GFP）下GFP的表达来观察对照组和所有神经元中缺乏nlr-1或仅缺乏多巴胺神经元的动物中的多巴胺神经元形态。我们注意到，在所有神经元中缺乏nlr-1的动物和多巴胺神经元中缺乏nlr-1的动物中，CEP神经元和轴突似乎在神经环束缚中存在缺陷（图2D，E和补充图2D，E）。

While approximately 65% of controls had well fasciculated CEP axons, only 30% of nlr-1 neuron knockout animals and 35% of nlr-1 dopamine knockout animals had well fasciculated axons (Fig. 2F, G). We also measured the distance between dopamine neuron axons in the nerve ring, defined as the distance between the top of the most anterior to the bottom of the most posterior axonal projection (Fig. 2D), and found it was greater in nlr-1 neuron knockouts and nlr-1 dopamine neuron knockouts compared to respective controls (Fig. 2H, I).

虽然大约65%的对照组具有束状的CEP轴突，但只有30%的nlr-1神经元敲除动物和35%的nlr-1多巴胺敲除动物具有束状的轴突（图2F，G）。。

Further, the volume of the dopamine axon projections was also higher in nlr-1 neuron knockouts and in nlr-1 dopamine neuron knockouts (Supplementary Fig. 2A–C). This suggests that nlr-1 plays a role in dopamine neuron structure and morphology. We then visualized the presynaptic morphology of dopamine neurons by expressing a mRuby tagged CLA-1, the C.

此外，在nlr-1神经元敲除和nlr-1多巴胺神经元敲除中，多巴胺轴突投射的体积也更高（补充图2A–C）。这表明nlr-1在多巴胺神经元的结构和形态中起作用。然后，我们通过表达mRuby标记的CLA-1（C）来可视化多巴胺神经元的突触前形态。

elegans orthologue of the presynaptic active zone marker bassoon (dat-1p::mRuby::cla-1). Control worms had.

突触前活动区标记巴松管（dat-1p：：mRuby：：cla-1）的线虫直系同源物。控制蠕虫。

nlr-1 is required for non-dopamine neuron mediated behavioral responses to dopamineTo further define the role of nlr-1 in dopamine signaling, we asked whether loss of nlr-1 in all neurons or in dopamine neurons alters responsiveness of the circuit to exogenous dopamine. As expected based on previous work29,31,33, control animals on food plus 10 mM dopamine significantly decreased their activity compared to animals on food alone (Fig. 3D).

非多巴胺神经元介导的多巴胺行为反应需要nlr-1为了进一步确定nlr-1在多巴胺信号传导中的作用，我们询问所有神经元或多巴胺神经元中nlr-1的缺失是否会改变电路对外源性多巴胺的反应性。正如根据以前的工作29,31,33所预期的那样，与单独食用食物的动物相比，食用食物加上10mM多巴胺的对照动物的活性显着降低（图3D）。

Animals lacking nlr-1 in all neurons again had significantly lower activity on food compared to controls, however they had no significant difference in activity on food compared to on food with dopamine (Fig. 3D). Meanwhile, animals lacking nlr-1 in dopamine neurons had a similar response to exogenous dopamine as controls (Fig. 3D).

与对照组相比，在所有神经元中缺乏nlr-1的动物对食物的活性再次显着降低，但是与含有多巴胺的食物相比，它们对食物的活性没有显着差异（图3D）。同时，多巴胺神经元中缺乏nlr-1的动物对外源性多巴胺的反应与对照组相似（图3D）。

This suggests that loss of nlr-1 in all neurons disrupts the ability to respond to dopamine, but loss of nlr-1 in dopamine neurons alone does not alter the downstream response to dopamine. These results further indicate that nlr-1 acts in non-dopamine neurons to facilitate the functional response to dopamine signaling, likely in downstream neurons.Pharmacologic manipulation of monoamine signaling confirms roles for nlr-1 in dopamine signalingTo determine how and where nlr-1 functions within monoamine and dopamine signaling, we utilized a modified setup to test the behavioral impact of pharmacologic agents and compounds that target monoamine and dopamine pathways.

这表明所有神经元中nlr-1的缺失都会破坏对多巴胺的反应能力，但仅多巴胺神经元中nlr-1的缺失并不会改变下游对多巴胺的反应。这些结果进一步表明，nlr-1在非多巴胺神经元中起作用，以促进对多巴胺信号传导的功能反应，可能在下游神经元中。单胺信号传导的药理学操作证实了nlr-1在多巴胺信号传导中的作用为了确定nlr-1在单胺和多巴胺信号传导中如何以及在何处起作用，我们利用改进的设置来测试靶向单胺和多巴胺途径的药物和化合物的行为影响。

Using a 96-well plate WormCamp (Worm Collective Activity Monitoring Platform) with populations of ~30 animals swimming in liquid in each well, we tested the impact of compounds on control and nlr-1 knockout strains (Supplementary Fig. 2F). We compared the impact of each compound on activity levels to activity levels of the respective genotype in ve.

使用96孔板WormCamp（蠕虫集体活动监测平台），每个孔中约有30只动物在液体中游泳，我们测试了化合物对对照和nlr-1敲除菌株的影响（补充图2F）。我们比较了每种化合物对ve中各基因型活性水平的影响。

nlr-1 contributes to localization/expression of the D2-like dopamine receptor dop-3

nlr-1有助于D2样多巴胺受体dop-3的定位/表达

To explore whether nlr-1 may be involved in dop-3 receptor localization or function, we obtained worms that express a GFP tagged DOP-3 under its endogenous promoter and utr (fosmid based), and compared expression in controls and nlr-1 neuron knockouts. We compared the number of neurons expressing DOP-3::GFP between controls and nlr-1 neuron knockout mutants and found nlr-1 knockouts had fewer DOP-3::GFP+ neurons than controls (Fig. 4F, G).

为了探索nlr-1是否可能参与dop-3受体的定位或功能，我们获得了在其内源性启动子和utr（基于fosmid）下表达GFP标记的dop-3的蠕虫，并比较了对照和nlr-1神经元敲除中的表达。我们比较了对照和nlr-1神经元敲除突变体之间表达DOP-3：：GFP的神经元数量，发现nlr-1敲除的DOP-3：：GFP+神经元比对照少（图4F，G）。

We then compared the expression of DOP-3::GFP in a putative command interneuron (AVER/L based on expression and location of the neuron), which was present in nearly all animals. We found that DOP-3::GFP covered less surface area in nlr-1 mutants (Fig. 4H), and the mean intensity of DOP-3::GFP was lower than controls (Fig. 4I).

然后，我们比较了DOP-3：：GFP在几乎所有动物中都存在的推定命令中间神经元（基于神经元的表达和位置的平均/L）中的表达。我们发现DOP-3：：GFP在nlr-1突变体中覆盖的表面积较小（图4H），并且DOP-3：：GFP的平均强度低于对照（图4I）。

These results suggest nlr-1 functions in dopamine receptor expression levels and localization, which may explain the reduced response to exogenous dopamine in animals lacking nlr-1 in all neurons. Collectively, these results demonstrate that nlr-1 has critical roles in the organization of dopamine circuits, controlling proper axonal localization and morphology and postsynaptic receptor localization, providing multiple potential mechanisms underlying the changes in dopaminergic circuit function and behavior we observed..

这些结果表明nlr-1在多巴胺受体表达水平和定位中起作用，这可能解释了所有神经元中缺乏nlr-1的动物对外源性多巴胺的反应降低。总的来说，这些结果表明nlr-1在多巴胺回路的组织中具有关键作用，控制适当的轴突定位和形态以及突触后受体定位，为我们观察到的多巴胺能回路功能和行为的变化提供了多种潜在的机制。。

nlr-1 regulates the food- and dopamine-dependent basal slowing response behaviorLastly, we wanted to determine whether nlr-1 contributes to other dopamine-mediated behaviors in C. elegans. The C. elegans basal slowing response behavior is when a well-fed animal encounters the edge of a food patch and undergoes a decrease in activity or slowing to remain on food, which is dependent on dopamine.

nlr-1调节食物和多巴胺依赖的基础减慢反应行为上，我们想确定nlr-1是否有助于秀丽隐杆线虫中其他多巴胺介导的行为。秀丽隐杆线虫的基础减慢反应行为是当一只喂养良好的动物遇到食物斑块的边缘并经历活动减少或减慢以保持食物，这依赖于多巴胺。

The slowing response behavior is quantified by comparing the number of body bends of animals that experimentally encounter food (animals placed on a plate with food) to animals that do not encounter food (animals placed on a plate with no food)(Fig. 5A, B). Control worms have significant reductions in body bends on food compared to off food.

通过比较实验性遇到食物的动物（放在有食物的盘子上的动物）和不遇到食物的动物（放在没有食物的盘子上的动物）的身体弯曲次数来量化减慢的反应行为（图5A，B）。与食物外相比，对照蠕虫在食物上的身体弯曲显着减少。

C. elegans lacking dopamine signaling, particularly through loss of dop-3 receptors, show a higher number of body bends on food compared to controls, with mixed findings as to if the number of body bends on food is significantly lower compared to body bends off food29,33,44. To test if loss of nlr-1 in dopamine neurons alters the basal slowing response, we performed this behavioral assay on the E.

C、 缺乏多巴胺信号传导的秀丽隐杆线虫，特别是通过失去dop-3受体，与对照组相比，食物上的身体弯曲次数更多，关于食物上的身体弯曲次数是否明显低于身体弯曲食物29,33,44。为了测试多巴胺神经元中nlr-1的丢失是否会改变基础减慢反应，我们在E上进行了这种行为测定。

coli strain HB101, where the difference in basal slowing response in dopamine mutants was previously shown, and OP50, the primary food source used in the behavioral assays included in this study. As previously reported, controls taken off food, washed, and placed on food have a significant reduction in body bends compared to controls taken off food, washed, and placed on plates without food.

大肠杆菌菌株HB101（先前已显示多巴胺突变体的基础减慢反应差异）和OP50（本研究中包括的行为测定中使用的主要食物来源）。如先前报道，与脱下食物，洗涤并放置在没有食物的盘子上的对照相比，脱下食物，洗涤并放置在食物上的对照显着减少了身体弯曲。

This behavioral response was the same in controls regardless of the food source encountered (Fig. 5C, D). cat-2 mutants, which lack dopamine, had a significant reduction in body bends on OP50 and HB101 compared to no food. However, cat-2 mutants did have signific.

无论遇到什么食物来源，这种行为反应在对照组中都是相同的（图5C，D）。缺乏多巴胺的cat-2突变体与不进食相比，在OP50和HB101上的身体弯曲显着减少。然而，cat-2突变体确实具有显着性。

C. elegans strains and cloningWorms were grown on plates with Nematode Growth Media (NGM) agar seeded with Escherichia coli OP50 bacteria as a food source and raised at 20 °C. Unless otherwise noted, the N2 Bristol strain was used as the control strain. Strains are listed in Supplementary Table 2. To generate tbh-1p::FLP, the tbh-1 promoter (forward primer: acattacttaaattaccctataaacttct, reverse primer: aagcggcgttgtttgtggtgcgcccgtaga) was subcloned to replace the unc-4 promoter in pKE42 (a kind gift of Kelsie Eichel) To generate dat-1p::cla-1::mRuby, the dat-1 promoter (forward primer: gcctattccagtatgacccctttgaagcag, reverse primer: ctgaaaacacatgaatctagtatagtttta) was subcloned to replace the unc-17 promoter in PK18154.

C、 线虫菌株和克隆虫在接种有大肠杆菌OP50细菌作为食物来源的线虫生长培养基（NGM）琼脂的平板上生长，并在20℃下培养。除非另有说明，否则使用N2 Bristol菌株作为对照菌株。。为了产生tbh-1p：：FLP，亚克隆tbh-1启动子（正向引物：ACATTACTTAAATTACCCTATAACTTCT，反向引物：AAGCGGCGTTGTGTGGTGCGCCCGTAGA）以替代pKE42中的unc-4启动子（Kelsie Eichel的一种礼物）以产生dat-1p：：cla-1：：mRuby，亚克隆dat-1启动子（正向引物：gcctattccagtatgacccctttgaagcag，反向引物：CTGAAAACACATGAATCTAGTAGTTTTA）以替代unc-17 PK18154中的启动子。

To generate dat-1p::cla-1::gfp, the dat-1 promoter (forward primer: gcctattccagtatgacccctttgaagcag, reverse primer: ctgaaaacacatgaatctagtatagtttta) was subcloned to replace the unc-17 promoter in PK06854. To generate dat-1p::unc-9(ΔN18), the dat-1 promoter (forward primer: gcctattccagtatgacccctttgaagcag, reverse primer: ctgaaaacacatgaatctagtatagtttta) was subcloned to replace the flp-13 promoter in pHM104 (flp-13p::unc-9(ΔN18)), a kind gift of Kota Mizumoto40.tph-1p::FLP strain: To generate plasmid pBN550 tph-1p::FLP for integration into universal MosSCI landing sites55 (a 2.3 kb tph-1p::FLP::glh-2 3’UTR fragment was excised from pBN28056 with XmaI and NotI and inserted into NgoMIV/NotI of pBN857.

为了产生dat-1p：：cla-1：：gfp，将dat-1启动子（正向引物：gcctattccagtatgacccctttgaagcag，反向引物：CTGAAAACACATGAATCTAGTAGTTTTA）亚克隆以取代PK06854中的unc-17启动子。为了产生dat-1p：：unc-9（ΔN18），将dat-1启动子（正向引物：gcctattccagtatgacccctttgaagcag，反向引物：CTGAAAACACATGAATCTAGTAGTTAGTTTTA）亚克隆以取代pHM104中的flp-13启动子（flp-13p：：unc-9（ΔN18）），这是Kota Mizumoto40的一种礼物。tph-1p：：flp菌株：产生质粒pBN550 tph-1p：：flp以整合到通用MosSCI着陆位点55（2.3kb TpP1）中用XmaI和NotI从pBN28056中切下H-1p：：flp：：glh-2 3'UTR片段，并将其插入pBN857的NgoMIV/NotI中。

Single-copy insertion of the tph-1p::FLP construct into the oxTi365 locus on chrV was done by microinjection into the gonads of EG8082 young adult hermaphrodites55,58. Plasmid pBN550 carrying FLP and unc-119(+) as selection marker was injected at 50 ng/µl together with pCFJ601 eft-3p::Mos159 (50 ng/µl), pBN1 lmn-1p::mCh::his-5857 (10 ng/µl), pCFJ90 myo-2p::mCh5.

通过显微注射到EG8082年轻成年雌雄同体的性腺中，将tph-1p：：FLP构建体单拷贝插入chrV上的oxTi365基因座55,58。将携带FLP和unc-119（+）作为选择标记的质粒pBN550与pCFJ601 eft-3p：：Mos159（50 ng/µl），pBN1 lmn-1p：：mCh：：his-5857（10 ng/µl），pCFJ90 myo-2p：：mCh5一起以50 ng/µl注射。

cla-1 presynaptic puncta analysisPuncta localization was analyzed using FIJI. Images were opened in FIJI, turned to z-stacks and saved in Adobe Photoshop, where the images were inverted, and the gamma was uniformly adjusted across all images to enhance contrast. The Z-stacks were loaded back into FIJI and thresholded uniformly such that synaptic puncta were visualized but background NLR-1 signal was not.

cla-1突触前斑点分析使用FIJI分析斑点定位。图像在斐济打开，转换为z堆栈并保存在Adobe Photoshop中，在那里图像被反转，并且在所有图像中均匀调整伽马以增强对比度。将Z堆栈加载回斐济并均匀阈值化，以使突触点可见，但背景NLR-1信号不可见。

The 3D-objects counter was then used to determine the number of puncta..

然后使用3D对象计数器确定点数。。

dop-3 receptor analysisdop-3 localization was assessed using strains with a fosmid-based GFP signal fused to the receptor (DOP-3:GFP) and analyzed in FIJI and Adobe Photoshop. Images were opened in FIJI, turned into z-stacks, then opened in Photoshop, and the number of GFP+ cell bodies was counted. The AVEL/R neuron was reliably expressed in nearly all images, so the surface area and average density was calculated for this neuron using FIJI’s object counter.Basal slowing responseWorms were tested for their basal slowing response as previously described29,33.

dop-3受体分析使用具有与受体融合的基于fosmid的GFP信号（dop-3:GFP）的菌株评估dop-3定位，并在FIJI和Adobe Photoshop中进行分析。图像在斐济打开，变成z堆栈，然后在Photoshop中打开，并计数GFP+细胞体的数量。AVEL/R神经元在几乎所有图像中都得到了可靠的表达，因此使用斐济的物体计数器计算了该神经元的表面积和平均密度。如前所述29,33，测试了基础减慢反应物的基础减慢反应。

Briefly, 5–8 worms were raised to day 1 young adults on OP50, washed using M9, and pipetted onto either plates coated with a ring of OP50 or HB101 or a non-seeded plate of NGM. The worms were allowed to acclimate for 5 min and then body bends were counted for each worm for 20 s. Worms were pipetted with enough distance between worms to reduce interactions, as interactions between worms altered their body bend number.

简而言之，将5-8条蠕虫在OP50上饲养至第1天的年轻成虫，用M9洗涤，然后移液到涂有OP50或HB101环的平板或NGM的非种子平板上。让蠕虫适应5分钟，然后对每只蠕虫的身体弯曲进行计数20秒。蠕虫之间的距离足够长，以减少相互作用，因为蠕虫之间的相互作用改变了它们的身体弯曲数量。

Worms on the seeded plates that did not reach the food after 5 min were not used.Statistics and reproducibilityFood deprivation WorMotel activity values were binned by hour to measure changes in foraging activity overtime and comparisons between activity of each genotype and condition at each time point were analyzed via two-way repeated measures ANOVA with a Tukey HSD post hoc test.

不使用5分钟后未到达食物的接种板上的蠕虫。统计和可重复性食物剥夺WorMotel活动值按小时分类，以测量超时觅食活动的变化，并通过双向重复测量ANOVA和Tukey HSD事后检验分析每个时间点每种基因型和条件的活动之间的比较。

A Greenhouse-Geisser correction was applied as sphericity was not assumed. The first 4 h of dopamine treatment WorMotels were aggregated and used for analysis, and differences between genotypes and conditions was analyzed via one-way ANOVA with a Tukey HSD post hoc test. The basal slowing response was analyzed using a one-way ANOVA with a Tukey HSD post hoc test.

由于未假设球形度，因此应用了Greenhouse-Geisser校正。多巴胺治疗的前4小时WorMotels被聚集并用于分析，基因型和条件之间的差异通过Tukey HSD事后检验的单因素方差分析进行分析。使用单因素方差分析和Tukey HSD事后检验分析基础减慢反应。

A Fischer’s exact test was conducted to compare the proportio.

进行了费歇尔精确检验以比较比例。

Data availability

数据可用性

All data that support the findings of this study are represented in the article, Supplementary tables, and figures and are available on Mendeley data as Hart, Michael (2024), “nlr-1/CNTNAP regulates dopamine circuit structure and foraging behaviors in C. elegans”, (doi: 10.17632/44g5zg4gt6.1). All data, plasmids, and strains are also available from the corresponding author upon request..

支持本研究结果的所有数据均在文章，补充表格和数字中表示，并可在Mendeley data上获得，如Hart，Michael（2024），“nlr-1/CNTNAP调节秀丽隐杆线虫的多巴胺回路结构和觅食行为”，（doi:10.17632/44g5zg4gt6.1）。所有数据，质粒和菌株也可应要求从通讯作者处获得。。

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G. & Buchner, A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav. Res. Methods 39, 175–191 (2007).Download referencesAcknowledgementsThe authors thank the Hart lab for technical assistance. The authors thank Dong Yan for providing NYL2730, which was used to cross nlr-1::mkate::frt into various strains and Peter Askjaer for providing BN1384 and BN1429, which were used to cross dat-1p::FLP::mNG and tph-1p::FLP into various strains, and for writing the methods for creation of BN1384.

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The authors thank Meera V. Sundaram and David M Raizen for their feedback on this project. The authors also thank Anthony D. Fouad (Tau Scientific) for technical support. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This work was supported by a Penn NGG Hearst Foundation Fellowship (B.L.B.), and by NIGMS of the NIH under 1R35GM146782 (M.P.H.).Author informationAuthors and AffiliationsDepartment of Genetics, University of Pennsylvania, Philadelphia, PA, 19104, USABrandon L.

作者感谢Meera V.Sundaram和David M Raizen对该项目的反馈。作者还感谢Anthony D.Fouad（Tau Scientific）的技术支持。一些菌株由美国国立卫生研究院研究基础设施项目办公室（P40 OD010440）资助的CGC提供。这项工作得到了宾夕法尼亚大学NGG赫斯特基金会奖学金（B.L.B.）和美国国立卫生研究院NIGMS根据1R35GM146782（M.P.H.）的支持。作者信息作者和附属机构宾夕法尼亚大学遗传学系，宾夕法尼亚州费城，19104，USABrandon L。

Bastien, William R. Haury, William R. Smisko & Michael P. HartAuthorsBrandon L. BastienView author publicationsYou can also search for this author in.

Bastien，William R.Haury，William R.Smisko＆Michael P.HartouthorsBrandon L.BastienView作者出版物您也可以在中搜索这位作者。

PubMed Google ScholarWilliam R. HauryView author publicationsYou can also search for this author in

PubMed谷歌学者William R.HauryView作者出版物您也可以在

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PubMed Google ScholarContributionsB.L.B. and M.P.H. conceived and designed the study and experiments and B.L.B. conducted all behavioral WorMotel experiments and basal slowing response experiments. M.P.H. designed and generated cloning, plasmids and injected to generate strains.

PubMed谷歌学术贡献b。五十、 B.和M.P.H.构思并设计了这项研究和实验，B.L.B.进行了所有行为WorMotel实验和基础减慢反应实验。M、 P.H.设计并产生克隆，质粒并注射以产生菌株。

W.R.H. and W.R.S. assisted with behavioral experiments. B.L.B. conducted microscopy experiments. B.L.B. processed and analyzed all WorMotel, basal slowing response, and microscopy data, B.L.B. and M.P.H. wrote the manuscript and all authors reviewed, revised, and approved the manuscript.Corresponding authorCorrespondence to.

W、 R.H.和W.R.S.协助进行行为实验。B、 L.B.进行了显微镜实验。B、 L.B.处理并分析了所有WorMotel，基础减慢反应和显微镜数据，B.L.B.和M.P.H.撰写了手稿，所有作者都审阅，修订并批准了手稿。。

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Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Asuka Takeishi and Benjamin Bessieres. A peer review file is available.

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Reprints and permissionsAbout this articleCite this articleBastien, B.L., Haury, W.R., Smisko, W.R. et al. nlr-1/CNTNAP regulates dopamine circuit structure and foraging behaviors in C. elegans.

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