RNA significantly attenuated HeLa cell proliferation. Furthermore, XAV939, an inhibitor of β‐catenin, was used to treat HeLa cells and the results demonstrated that HMQ‐T‐F2 inhibited proliferation and migration via the inhibition of the Wnt/β‐catenin pathway.Looking for novel, effective and less toxic therapies for cervical cancer is of significant importance. In this study, we reported that HMQ‐T‐F2(F2) significantly inhibited cell proliferation and transplantable tumour growth. Mechanistically, HMQ‐T‐F2 inhibited HeLa cell growth through repressing the expression and nuclear translocation of β‐catenin, enhancing Axin expression, as well as downregulating the Wnt downstream targeted proteins. Knock‐down of a checkpoint β‐catenin by si Chemotherapy is often used to treat tumours that have spread or have reappeared after treatment.22.12 atmosphere with saturated humidity.HMQ‐T‐F2 (purity > 98%) were synthesized in the Research and Engineering Center for Natural Medicine, Xi'an Jiaotong University. ZR‐75‐30, MCF‐7, SMMC‐7721, Hep3B, Bel‐7402, A549, A431, ECV304, LOVO, and human cervical cancer cell line HeLa, Ca Ski, SiHa and MS751 were obtained from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences. Human normal cervical epithelial cells were purchased from CHI Scientific, Inc  and grown in DMEM/F12 medium with 10% (v/v) FBS and ECGS, Hydrocortisone, Heparin supplements. ZR‐75‐30, Hep3B, ECV304 and LOVO cell lines were cultured in DMEM medium with 10% (v/v) FBS; MCF‐7, SMMC‐7721, Bel‐7402, Ca Ski and HeLa cells were cultured in RPMI‐1640 medium with 10% (v/v) FBS; A431 and A549 cells were grown in F12 medium with 10% (v/v) FBS; SiHa and MS751 cells were cultured in MEM medium with 10% (v/v) FBS. All cell lines were incubated at 37°C in a 5% COFour to six week old NOD/SCID nude male mice were purchased from Hunan SJA Laboratory Animal Co., Ltd.  and housed in the Experimental Animal Center of Xi'an Jiaotong University.2.2ZR‐75‐30, MCF‐7, SMMC‐7721, Hep3B, Bel‐7402, A549, A431, ECV304, LOVO, Ca Ski, SiHa, MS751, HeLa and human normal cervical epithelial cells were seeded and cultured with HMQ‐T‐F2 for 48 hours. Cells were cultured with MTT and analysed using a microplate reader .2.3HeLa cells were seeded and treated with HMQ‐T‐F2 for 48 hours. The colonies were stained with crystal violet and photographed.2.46 HeLa cells. Mice were randomly assigned and respectively treated with vehicle (0.5% CMC‐Na), HMQ‐T‐F2 . The relative mRNA expression levels were calculated by the comparative 22.6After treatment, HeLa cells were collected and lysed in RIPA buffer. The samples were separated by SDS‐PAGE electrophoresis.2.7After incubation with HMQ‐T‐F2 for 48 hours, HeLa cells were fixed and incubated with a primary antibody against β‐catenin (1:200) at 4°C overnight. Then, cells were incubated with an FITC‐labelled anti‐rabbit secondary antibody (1:50) for 1 hour. The nucleus was stained with DAPI. Fluorescent signals were detected using an inverted fluorescence microscope .2.8Specific knockdown was achieved using a double‐stranded siRNAs against β‐catenin and nonspecific siRNA which were obtained from Shanghai GenePharma Co., Ltd . Transfected cells were subjected to RT‐PCR and western blotting. The transfected cells were used for cell proliferation assay.2.9P value <.05 was considered statistically significant. *P < .05, **P < .01, ***P < .001 compared to control group, #P < .05, ###P < .001 compared to HMQ‐T‐F2‐treated groups, &&&P < .001 compared to XAV939‐treated groups. Data are expressed as means ± SEM.One‐way analysis of variance (ANOVA) and further Tukey's multiple comparison test as well as independent‐samples t test were used. A 3Chemotherapy plays a vital role in treating cervical cancer that has spread or has relapsed after treatment.β‐catenin, a central mediator in the Wnt/β‐catenin signalling pathway, is expressed in many solid tumours.The stability of the Wnt pathway transcription factor β‐catenin is tightly regulated by the multi‐subunit destruction complex. The β‐catenin destruction complex, which consists of Axin, APC, CK1 promotes proteasome‐mediated proteolysis mediated proteolysis of phosphorylatedβ‐catenin.In summary, this study demonstrated that HMQ‐T‐F2 inhibited cervical HeLa cell proliferation in vitro and in vivo. The mechanism underlying these effects involves β‐catenin degradation and maintenance of Axin stability by HMQ‐T‐F2 and therefore, the inhibition of the Wnt/β‐catenin signalling pathway. This study suggests that HMQ‐T‐F2 potentially have a therapeutic value for cervical cancer treatment.The authors declare that they have no conflict of interests to disclose.BLD, TFY and YJM performed the main experiments and summarized the results. NM and XPS assisted in performing the experiments. DDZ assisted in interpreting the data. JZ supplied the compound. BLD wrote the manuscript. BLD and YMZ provided the concept, funding, supervision and assisted in writing the manuscript. All authors read and approved the final manuscript.


C. elegans oscillators remains unknown. Through cell ablation, electrophysiology, and calcium imaging, we show: (1) forward and backward locomotion is driven by different oscillators; (2) the cholinergic and excitatory A-class motor neurons exhibit intrinsic and oscillatory activity that is sufficient to drive backward locomotion in the absence of premotor interneurons; (3) the UNC-2 P/Q/N high-voltage-activated calcium current underlies A motor neuron’s oscillation; (4) descending premotor interneurons AVA, via an evolutionarily conserved, mixed gap junction and chemical synapse configuration, exert state-dependent inhibition and potentiation of A motor neuron’s intrinsic activity to regulate backward locomotion. Thus, motor neurons themselves derive rhythms, which are dually regulated by the descending interneurons to control the reversal motor state. These and previous findings exemplify compression: essential circuit properties are conserved but executed by fewer numbers and layers of neurons in a small locomotor network.Cell- or network-driven oscillators underlie motor rhythmicity. The identity of  Central pattern generators (CPGs) are rhythm-generating neurons and neural circuits with intrinsic oscillatory activities. Across the animal phyla, CPGs underlie rhythm of motor behaviors that are either continuous, such as breathing and heartbeat, or episodic, such as chewing and locomotion . CPGs thThe concept of locomotor CPGs originated from the observation that decerebrate cats could sustain rhythmic hind limb muscle contraction . In deafC. elegans  that drive MN activity . In vertKiehn, 2006 elegans  and zebr elegans  in a conC. elegans generates rhythmic and propagating body bends that propel either forward or backward locomotion. Synaptic wiring of the adult locomotor system has been depicted by serial electron microscopy  that underlies their oscillation, or, through manipulating the synaptic inputs from their descending premotor INs AVA, alter the velocity, propensity and maintenance of the reversal motor state. We conclude that the C. elegans motor circuit is a CPG-driven locomotor network, except that its rhythm is derived from excitatory MNs.Here, we addressed these questions through cell ablation, electrophysiology, genetics, and calcium imaging. We reveal that C. elegans neurons to date do not fire classic action potentials  support the notion that compression - a single neuron or neuron class assuming the role of multiple layers in complex neural circuits - is a general property of small circuits. This property allows small nervous systems to serve as compact models to dissect the organizational logic of neural circuits, exemplified by the intricate roles of a mixed synapse configuration conserved not only at the locomotory, but also other motor circuits (see Discussion).C. elegans oscillators are present, we first examined the behavioral consequence of ablating MNs or premotor INs. In previous studies, ablation was restricted to a few neurons and in a small number of animals . Strikingly, A-MNs drive backward locomotion with robust bending in the absence of premotor INs , implyinBecause A-MNs can organize backward movements without premotor INs, they may function as either a chain of phase-locked oscillators, or as flexors, where each neuron is sequentially activated by bending of its neighboring body segment. These possibilities can be distinguished by examining the effect of sparse removal of A-MNs. In the former case, the removal of individual A-MNs should alter, but not prevent, bending propagation during backward locomotion. In the latter case, bending propagation shall stall at most posterior body segments that the ablated A-MNs innervate.Our ablation results ; Video 3Quantitatively, ablation of A-MNs from either the anterior, mid-body, or posterior segments  caused sCPGs should exhibit self-sustained oscillatory activity. We sought experimental evidence for such a property by performing electrophysiology and calcium imaging analyses on A-MNs.n = 10), we observed periodic rhythmic PSC (rPSC) bursts (n = 10), we observed PSC bursts of similar characteristics, but as sporadic single-unit events. These results suggest that A-MNs generate periodic electrical activities without premotor INs, and premotor INs may inhibit A-MN rhymthic activity.First, we examined a dissected neuromuscular preparation consisting of an exposed ventral nerve cord and the body wall muscles that they innervate . The maj) bursts  at ~90 s) bursts . The rPSn = 11).In the current clamp configuration, we observed periodic action potential bursts that corresponded to the rPSC bursts after premotor INs and B-MNs were ablated . Muscle CPGs exhibit oscillatory activity in the presence of sustained inputs. We further examined the effect of direct stimulation of excitatory MNs by a light-activated rhodopsin Chrimson . In thesResults from A-MN calcium imaging in intact animals also support their oscillatory property. To reduce potential effects of proprioceptive coupling, we recorded the A-MN activity from intact animals that were fully immobilized using the surgical glue. While sporadic calcium activities were occasionally observed for some A-MNs in non-ablated wildtype control animals, robust calcium oscillation was revealed in A-MNs in all animals after the co-ablation of premotor INs and B-MNs . IndividLastly, we performed simultaneous calcium imaging of a cluster of posteriorly positioned A-MNs in moving animals, after we removed all premotor INs and B-MNs . ConsistC. elegans, similar to what has been reported for the spinal cord preparations versus immobilized or mobile vertebrates  allele for the pore-forming α-subunit of the L-VGCC CaV1α EGL-19  mutants  animals, after co-ablating premotor INs and B-MNs. Devoid of premotor IN inputs, wildtype DA9 exhibited periodic calcium oscillations (unc-2(lf) mutants, both the amplitude and frequency of these oscillations were severely reduced ). Restoration of UNC-2 in A-MNs alone mutants was sufficient to restore oscillation of DA9’s calcium signals in unc-2(lf) mutants  near null mutants, when premotor INs and B-MNs were ablated, DA9 exhibited periodic calcium oscillations as in wildtype animals . Indeed, in addition to the presynaptic localization along the neuronal processes in both the central  mutants exhibited drastically reduced A-MN- dependent rPSC bursts (unc-2 gain-of-function (gf) mutations  that reduce the channel inactivation kinetics, resulting in prolonged channel opening . In contrast to the case of unc-2(lf) mutants, upon ablation of premotor INs and B-MNs, unc-2(gf) exhibited endogenous rPSC bursts with strikingly higher frequency and amplitude than wildtype animals (unc-2(gf) animals when compared to wildtype animals (unc-2(lf) mutants, specific restoration of wildtype UNC-2(WT) or Gain-of-function UNC-2(GF) in A-MNs was sufficient to rescue the frequency and amplitude of DA9’s oscillation  and unc-2(gf) mutants exhibited substantially reduced and increased reversal velocities, respectively, than ablated wildtype animals  mutants (unc-7(lf) mutants, despite the presence of AVA (unc-7(lf) mutants (unc-13(lf) (unc-7(lf) mutants  , and unc mutants , confirmunc-7 mutants display a kinker posture and drastically increased spontaneous reversals, in contrast to wildtype animal’s strong bias for forward locomotion , but not unc-13(lf) mutants in which synaptic transmission was abolished (unc-2(lf) – simultaneously rescuing their oscillation and NMJ activities – restores backward locomotion in the absence of premotor INs.In addition to a role in exocytosis, the P/Q/N-type calcium conductance produces A-MN membrane oscillation. In bolished , A-MN caAn exocytosis-independent role of high-voltage-activated calcium channels in membrane oscillation has been noted in vertebrates. In isolated lamprey spinal neuron somata, the N-type calcium currents prominently potentiate action potential bursts, and are coupled with calcium-activated potassium currents that terminate the bursts . The intC. elegans is superficially at odds with several fundamental features of CPG-driven networks: its genome does not encode voltage-activated sodium channels that typically drive action potential bursts, and all examined neurons to date, MNs and premotor INs included, are non-spiking. Instead, MNs and premotor INs exhibit plateau potentials upon stimulation  plates seeded with OP50, and maintained at 15°C or 22°C. Unless stated otherwise, the wildtype animal refers to the Bristol N2 strain. Strains that contain optogenetic transgenes  were cultured in darkness on NGM plates supplemented with or without ATR  were maintained in darkness on NGM plates.The complete lists of constructs, transgenic lines and strains generated or acquired for this study are provided in hout ATR . Strainsunc-2 was identified in a suppressor screen for the 'fainter' motor defects of unc-80(e1069) mutants. Both e1069; hp647 and hp647 animals exhibit hyperactive locomotion with high movement velocity and frequent alternation between forward and backward locomotion (shuttling) . hp647 was mapped between egl-17 and lon-2 on the X chromsome. Co-injected fosmids WRM0628cH07 and WRM0616dD06 rescued the shuttling phenotype exhibited by hp647 and reverted unc-80(e1069); hp647 to unc-80(e1069)-like fainter phenotypes. Subsequent exon and genomic DNA sequencing revealed a causative, gf L653F mutation in unc-2(hp647). unc-2(hp858) is an insertion allele where GFP was fused immediately in front of the ATG start codon of the unc-2 locus by CRISPR. hp858 animals exhibit wildtype motor behaviors. Other genetic mutants used for constructing transgenic lines and compound mutants were obtained from the Caenorhabditis Genetics Center (CGC); all were backcrossed at least four times against N2 prior to analyses.C. elegans genomic DNA. Promoters include the 5.1 kb Pnmr-1, 4.8 kb Prig-3, 2.8 kb Psra-11, 1.8 kb Pacr-2s, 2.5 kb Punc-4, 4.2 kb Pacr-5, 0.9 kb Pttr-39, 2.8 kb Pceh-12, 2.7 kb Punc-129(DB), and 0.86 kb Plgc-55B genomic sequence upstream of the respective ATG start codon. The Pnmr-1 used in this study excluded a 2 kb internal fragment that encodes cex-1, which interferes with reporter expression  and OZM2302 (AAGGTACCGATGCGTTGAAGCAGTTTC CC) using pBALU14 as the template. Two sets of bicistronic expression reporters used in this study, codon-optimized mCherry and EBFP, were gifts of Desai  and Calarco (University of Toronto), respectively. They were used for behavioral analyses and to be combined with calcium imaging analyses, respectively.For neuronal ablation constructs, MiniSOG  fused wiPrig-3 was used to drive the expression of a UNC-7::GFP cassette, and Pnmr-1 was used to drive FLP expression.The FRT-FLP system was used to generate two constructs that are co-injected to achieve AVA-specific UNC-7 expression. hpEx) were generated by co-injecting an injection marker with one to multiple DNA construct at 5–30 ng/μl. Animals that carry integrated transgenic arrays (hpIs) were generated from the hpEx animals by UV irradiation, followed by outcrossing against N2 at least four times. L4-stage or young adults (24 hr post L4) hermaphrodites were used in all experiments.Transgenic animals that carry non-integrated, extra-chromosomal arrays  and a few other unidentified neurons, respectively . On platA single 12–18 hr post-L4 stage adult hermaphrodite, maintained on standardculture conditions, was transferred to a 100 mm imaging plate seeded with a thin layer of OP50. One minute after the transfer, a 2-minute video of the crawling animal was recorded on a modified Axioskop 2  equipped with an automated tracking stage MS-2000  and a digital camera .C. elegans were recorded using an in-house developed automated tracking program. All images were captured with a 10X objective at 10 frames per second. Data recorded on the same plate and on the same day were pooled, quantified and compared.Imaging plates were prepared as follows: a standard NGM plate was seeded with a thin layer of OP50 12–14 hr before the experiment. Immediately before the transfer of worms, the OP50 lawn was spread evenly across the plate with a sterile bent glass rod. Movements exhibited by Post-imaging analyses utilized an in-house developed ImageJ Plugin  and in-hDA9 recording from immobilized intact animals  in moving animals  was perfPunc-4 promoter exhibits strongest expression in A-MNs.In both preparations, GCaMP and RFP signals were simultaneously acquired using the Dual-View system , and the GCaMP/RFP ratios were calculated to control for motion artifacts and fluorescence bleaching during recording. Late L4 stage animals were used for all calcium imaging experiments, when the hpIs583 (A- and B-MNs miniSOG) or YX167 hpIs366/qhIs4; qhIs1 (A-MNs miniSOG) L2 larva were immobilized on a 4% agar pad with 5 mM tetramisole. Region-specific illumination was performed by targeting a 473 nm laser at an arbitrary portion of the animal using a digital micromirror device (DMD) through a 20X objective  by a pulsed infrared laser illumination system (Most posterior MNs (VA12-DA9) ablation was also performed in YX148 n system  modifiedFor behavior recordings, each animal was transferred to an unseeded NGM plate, and on plate crawling was recorded for at least 5 min under bright field illumination. If the animal became sluggish or idle, the plate was agitated using the vibration motor from a cell phone. After recordings, each animal was imaged at 40X for RFP pattern for the entire body and we manually assigned present and missing neurons based on their relative positions and commissural orientation . For YX1Analyses of locomotion of ablated and control animals were carried out using WormLab  and in-house Matlab codes. Data from all three ablation methods were pooled to generate the summary statistics. Bouts of reversals that lasted at least 3 s were analyzed for the speed of wave propagation. Curvature segmentations from the behavioral recordings were constructed using WormLab . Wave speed was measured as a function of body coordinate and time, by taking the derivative of each curvature map with respect to time (dκ/dt), and to body coordinate (dκ/dC). Wave speed was defined as the ratio between these gradients (body coordinate/s). Wave speed was averaged over the length of each bout, and binned for the anterior (5–25% of body length from the head), mid-body (40–60%), and posterior (75–95%) region in each bout.2 0.1; MgCl2 5; BAPTA 1; HEPES 10; Na2ATP 5; Na2GTP 0.5; cAMP 0.5; cGMP 0.5, pH7.2 with KOH,~320 mOsm. cAMP and cGMP were included to maintain the activity and longevity of the preparation. The bath solution consists of (in mM): NaCl 150; KCl 5; CaCl2 5; MgCl2 1; glucose 10; sucrose 5; HEPES 15, pH7.3 with NaOH,~330 mOsm. Chemicals and blockers were obtained from Sigma unless stated otherwise. Experiments were performed at room temperatures (20–22oC).Dissection and recording were carried out using protocols and solutions described in , which w2) for hpIs166 and hpIs279, and at 625 nm (from 1.1 mW/mm2), for hpIs569 and hpIs578, respectively, controlled by the Axon amplifier software. One-second light exposure, a condition established by our previous study , Clampfit (Molecular Devices), Image J , R (http://www.R-project.org.), Matlab , and Excel . For electrophysiology and calcium imaging, unless specified otherwise, each recording trace was obtained from a different animal; data were presented as the mean ± SEM.The Mann-Whitney U test, two-tailed Student’s  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Eve Marder (Senior Editor) and three reviewers, one of whom, Ronald L Calabrese (Reviewer #1), is a member of our Board of Reviewing Editors. The following individuals involved in review of your submission have agreed to reveal their identity: Akinao Nose (Reviewer #2); David Biron (Reviewer #3).Thank you for submitting your article "Excitatory Motor Neurons are Local Central Pattern Generators in an Anatomically Compressed Motor Circuit for Reverse Locomotion" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:C. elegans model system, but has real general interest as well. It dissects the motor system of C. elegans by selective cell ablation and mutant analysis and uses behavior and electrophysiology as assays to identify and define the reverse locomotion "CPG". While CPG may not be the right term in the minds of many, the authors do indeed identify a source of endogenous oscillation in motor neurons that underlies rhythmic reverse locomotion. For reverse locomotion the conclusion must be that the A-MNs, which are known to be dedicated to reverse locomotion, form a chain of motor neuron oscillators. The implication is that the B-MNs motor neurons, which are known to be dedicated to forward locomotion, form a similar chain of oscillators, but it is not nailed down completely and a head oscillator certainly exists that is not characterized. The limitation of the detailed analysis to reverse locomotion is not a real drawback, however, because the analysis uncovers important properties of the reverse locomotion circuit. Membrane potential oscillations are supported by HVA Ca channels (UNC-2), and very interestingly, premotor interneurons are not necessary for reverse (or forward) locomotion but exert state dependent control over A-MN oscillations and reverse locomotion initiation and maintenance. A-type-INs make mixed heterotypic and rectifying gap junction and excitatory chemical synapses on A-MNs to exert this control. At rest, AVA-A gap junctions dampen the excitability of coupled premotor INs and oscillatory activity of MNs to reduce propensity for reversals. Upon activation, AVA potentiate AMNs predominantly through excitatory chemical synapses, with a minor contribution from gap junctions. This is a truly interesting and important finding and should be more front and center in Discussion.This is a very interesting manuscript that is very important for the The work is very carefully and thoroughly done with appropriate controls, and the data analyzed appropriately. Necessary data appear in figures in an easily accessible form.Essential revisions:1) Please explain why B-MNs, in addition to premotor INs, have to be ablated for the A-MN to show intrinsic rhythmic activity . If the 2) Subsection “A-MNs exhibit oscillatory activity independent of premotor IN inputs”, last paragraph: The authors state that all A-MNs show oscillating activity but with differences in the intensity. However, the authors only show results on DA9 in the figure . Can oth3) The full distributions of velocities e.g.,  are usef4) Subsection “A-MNs exhibit oscillatory activity independent of premotor IN inputs”, fourth paragraph: the data in Figure 5—figure supplement 1 is interpreted without much justification. Why did peaks in A-20% qualify as rPSC bursts while peaks in B-1% didn't? What were the criteria?5) Subsection “A-MNs exhibit oscillatory activity independent of premotor IN inputs”, last paragraph: calcium imaging was performed in L4 larvae as opposed to measuring rPSCs in adult preps. Was this due to body thickness? Could development have contributed to the difference in the frequencies? Is there a difference in reversal behavior between L4 and adult B-MN / INs ablated worms?6) The Results section "UNC-2 is an endogenous constituent of A-MN oscillation" is not sufficiently clear regarding the calcium flows. The second paragraph states that loss of function of the VGCC UNC-2 severely reduces calcium oscillations. Prior to explaining unc-7 mutants should exhibit increased reversal speed and/or persistence. Unfortunately, unc-7 behavioral  data. The optogenetic experiment may well be too onerous. Scoring reversal speed/persistence for an existing strain or explaining why the simple prediction is wrong may fall within the scope of this work.7) A simple prediction of the model is that in the absence of B-MNs/INs or if AVA is optogenetically activated, 8) "[P]ersistent body bends in premotor IN-less animals mainly originated from A-MN activity." 9) The manuscript does not comment on the ~50s calcium oscillation cycle in A-MNs. Does it correspond to some behavioral timescale or have a hypothesized significance? Do the mean velocities seem related? It may be the case that there is not much to say on the subject.10) The writing needs reconsideration. Much of the important information is in the figure legends and the paper is thus tedious to follow. Putting more description of the figure in the text would help. The figures are very complicated and the data could be separated out into smaller figures. Word choice is not always the best and led to confusion for this reviewer. Many detailed examples of words that need clarifying are made on the manuscript pdf in Discussion and Introduction. The kymograms could be better explained so that readers unfamiliar with them can understand what the complex patterns in these kymograms from ablated and mutant animals mean.C. elegans with vertebrate spinal cord or even invertebrate networks like that for leech swimming will be a hard sell to a veteran CPG person like myself. To my mind this is not the real interest of the paper. There are two major impacts arising from this work. 1) You have solved a pressing problem in C. elegans neurobiology which has plagued the field for over a decade. This should be emphasized. 2) You have made a truly brilliant discovery of the state dependent regulation of A-MN oscillations by the conjoint electrical and chemical synapses of premotor interneurons. This should be a centerpiece of the paper. The ideas on circuit compression are not compelling. Is the circuit compressed in C. elegans or is it expanded by evolution in leech? The first neurons in evolution were probably multifunctional (sensory-motor) and then evolution led to proliferation of neurons and specialization. That invertebrate neurons are very multifunctional has to be a truism since I was a graduate student and that motor neurons can be oscillatory has also been known as long. These arguments should be deemphasized.11) The concept of CPG is not well articulated and its meaning is stretched a bit. You are really dealing with individual neuronal oscillators and not CPGs and equating the structure of the reverse locomotion circuit in 12) Title could be shortened and refocused to match the revised Discussion. Summary:This is a very interesting manuscript that is very important for the C. elegans model system, but has real general interest as well. It dissects the motor system of C. elegans by selective cell ablation and mutant analysis and uses behavior and electrophysiology as assays to identify and define the reverse locomotion "CPG". While CPG may not be the right term in the minds of many, the authors do indeed identify a source of endogenous oscillation in motor neurons that underlies rhythmic reverse locomotion.Thank you. We are pleased to make contributions to our understanding on how motor circuits operate. We agree that in most context of this study, oscillator is a more suitable choice. We have made these changes, and are grateful for the suggestion.For reverse locomotion the conclusion must be that the A-MNs, which are known to be dedicated to reverse locomotion, form a chain of motor neuron oscillators. The implication is that the B-MNs motor neurons, which are known to be dedicated to forward locomotion, form a similar chain of oscillators, but it is not nailed down completely and a head oscillator certainly exists that is not characterized. The limitation of the detailed analysis to reverse locomotion is not a real drawback, however, because the analysis uncovers important properties of the reverse locomotion circuit.Indeed. Although we chose to focus on oscillators for the reverse motor circuit, we could not address it without first knowing that different oscillators underlie forward and reverse movements . This coAnother critical piece of information from Such a distinction alludes to a potential difference in the functional relationships between the two classes of motor neurons with their respective input interneurons. More important is the notion that only when the A-MNs activity was removed could the intrinsic property of the B-MNs to be addressed in isolation . EstabliC. elegans motor circuit.We had chosen to focus on the reverse motor circuit for two reasons: 1) examining the functional relationship between A motor neurons and the descending premotor interneurons has been a long-standing goal of our group; 2) we would like to collaborate with and help other groups who are interested in the Membrane potential oscillations are supported by HVA Ca channels (UNC-2), and very interestingly, premotor interneurons are not necessary for reverse (or forward) locomotion but exert state dependent control over A-MN oscillations and reverse locomotion initiation and maintenance. A-type-INs make mixed heterotypic and rectifying gap junction and excitatory chemical synapses on A-MNs to exert this control. At rest, AVA-A gap junctions dampen the excitability of coupled premotor INs and oscillatory activity of MNs to reduce propensity for reversals. Upon activation, AVA potentiate AMNs predominantly through excitatory chemical synapses, with a minor contribution from gap junctions. This is a truly interesting and important finding and should be more front and center in Discussion.Thank you. We have revised the Abstract, Introduction, and Discussion to emphasize this finding. Further, we present additional calcium imaging and behavioural results that support the state-dependent dual-regulation model  Please explain why B-MNs, in addition to premotor INs, have to be ablated for the A-MN to show intrinsic rhythmic activity . If the Sorry for the confusion. A-MN’s intrinsic activity does not depend on the presence or absence of B-MNs; B-MNs only interfere with their propagation after premotor INs are removed. In The behavioural phenotype of premotor IN ablated animals , either alone or in combination with A-MNs or B-MNs  indicateConsistent with this notion, we performed the complementary experiment. In preparations where we removed A-MNs along with premotor INs, the mini frequencies were drastically reduced, but the rPSC burst was not increased . These r2) Subsection “A-MNs exhibit oscillatory activity independent of premotor IN inputs”, last paragraph: The authors state that all A-MNs show oscillating activity but with differences in the intensity. However, the authors only show results on DA9 in the figure . Can othIn We thought that the phasic relationships among multiple A-MNs should be analyzed in moving animals. This was why we performed simultaneous calcium imaging data for three A-MNs  in animals where we removed all premotor INs and B-MNs, and when animals were moving  The full distributions of velocities e.g.,  are usefThank you for this helpful suggestion. We added mean velocities and deviations in figures where they help to clarify their effect on animals’ motility  or veloc4) Subsection “A-MNs exhibit oscillatory activity independent of premotor IN inputs”, fourth paragraph: the data in Figure 5—figure supplement 1 is interpreted without much justification. Why did peaks in A-20% qualify as rPSC bursts while peaks in B-1% didn't? What were the criteria?Sorry for the confusion. We applied the criteria that were defined in a previous study . Briefly, rPSC bursts consisted of a train of depolarization events , which is distinct from the high-frequency monophasic and multiphasic mPSC events . Events in Figure 5—figure supplement 1 (B-1%) fell in the second category .5) Subsection “A-MNs exhibit oscillatory activity independent of premotor IN inputs”, last paragraph: calcium imaging was performed in L4 larvae as opposed to measuring rPSCs in adult preps. Was this due to body thickness? Could development have contributed to the difference in the frequencies? Is there a difference in reversal behavior between L4 and adult B-MN / INs ablated worms?We used L4 animals for calcium imaging analyses for two reasons: 1) we used L4 animals for behavioural analyses; 2) the Punc-4 promoter that we used to express the calcium sensor is strong in A-MNs in L4, but falls off sharply afterwards, making recording in adults more noisy. We don’t think that the developmental difference leads to the two-fold difference of the frequency of rPSC and calcium oscillation. In a few DA9 calcium imaging recordings that we obtained in young adults , they ex6) The Results section "UNC-2 is an endogenous constituent of A-MN oscillation" is not sufficiently clear regarding the calcium flows. The second paragraph states that loss of function of the VGCC UNC-2 severely reduces calcium oscillations. Prior to explaining Thank you. We have revised this section accordingly.Finally, could the authors comment about the origin of the calcium they observe? The key role of UNC-2 seems to suggest that it is predominantly extracellular. Is this a valid conclusion?These results are indeed consistent with UNC-2 being the origin of extracellular calcium. We did not make an explicit claim previously because of the possibility that release from the intracellular calcium store, upon such an extracellular entry, also contribute to the observed calcium profile in DA9 soma. We have now clarified this point .7) A simple prediction of the model is that in the absence of B-MNs/INs or if AVA is optogenetically activated, unc-7 mutants should exhibit increased reversal speed and/or persistence. Unfortunately, unc-7 mutants, results from which further validate the hypothesis that AVA premotor INs inhibit A-MN activity through gap junctions to reduce propensity for the reverse motor state.Thank you for these suggestions. We performed new experiments on unc-7 mutants  "[P]ersistent body bends in premotor IN-less animals mainly originated from A-MN activity." Sorry about the lack of clarity. We reworded relevant sections in the revised manuscript; a more detailed explanation follows.In the absence of premotor INs, animals continued to exhibit body bends . When weBut in the absence of premotor interneurons, B-MNs strongly antagonize A-MN-driven reverse movements. Because B-MNs are activated by body bending , a simple interpretation is that in the absence of premotor INs, body bending primarily results from the high intrinsic A-MNs, which may subsequently potentiate B-MNs that reside in the body segment to interfere with their propagation.Similarly, While the ‘pause’  describeWe agree with the reviewer that these behaviour results suggest that there is no equivalent independent reversal oscillator in the tail. Consistent with this notion, animals without premotor INs or MNs could exhibit high frequency oscillation of the head, but not of the tail .We refrained from elaborating on these hypotheses because they suggest complex, yet-to-be addressed relationships between the forward and reversal oscillators. We hope to resolve it in future studies.9) The manuscript does not comment on the ~50s calcium oscillation cycle in A-MNs. Does it correspond to some behavioral timescale or have a hypothesized significance? Do the mean velocities seem related? It may be the case that there is not much to say on the subject.In our calcium imaging experiments on crawling animals, after the ablation of premotor INs/B-MNs, they exhibited ~10s reversal undulation cycle on plates  The writing needs reconsideration. Much of the important information is in the figure legends and the paper is thus tedious to follow. Putting more description of the figure in the text would help. The figures are very complicated and the data could be separated out into smaller figures. Word choice is not always the best and led to confusion for this reviewer. Many detailed examples of words that need clarifying are made on the manuscript pdf in Discussion and Introduction. The kymographs could be better explained so that readers unfamiliar with them can understand what the complex patterns in these kymographs from ablated and mutant animals mean.Thank you for the careful, constructive editorial comments. We thrive to make the description and illustration of ours and other’s work precise and clear. We have incorporated suggested editions, provided more descriptions for behavioural analyses, and reorganized figures to convey key messages.11) The concept of CPG is not well articulated and its meaning is stretched a bit. You are really dealing with individual neuronal oscillators and not CPGs and equating the structure of the reverse locomotion circuit in C. elegans with vertebrate spinal cord or even invertebrate networks like that for leech swimming will be a hard sell to a veteran CPG person like myself. To my mind this is not the real interest of the paper.C. elegans motor rhythm is CPG-driven is important for C. elegans motor circuit, but we agree with you that ‘oscillator’ is a more appropriate word to define A-MNs’ activity.We appreciate these comments. In the previous manuscript, we indeed did not make sufficient distinction between the usage of ‘CPG’ and ‘oscillator’. This in part reflects where the field was: the presence of oscillators was debated. Establishing that There are two major impacts arising from this work. 1) You have solved a pressing problem in C. elegans neurobiology which has plagued the field for over a decade. This should be emphasized. 2) You have made a truly brilliant discovery of the state dependent regulation of A-MN oscillations by the conjoint electrical and chemical synapses of premotor interneurons. This should be a centerpiece of the paper. The ideas on circuit compression are not compelling. Is the circuit compressed in C. elegans or is it expanded by evolution in leech? The first neurons in evolution were probably multifunctional (sensory-motor) and then evolution led to proliferation of neurons and specialization. That invertebrate neurons are very multifunctional has to be a truism since I was a graduate student and that motor neurons can be oscillatory has also been known as long. These arguments should be deemphasized.Thank you for the compliment to our work. We have revised the title, Abstract, Introduction, figures, and Discussion to emphasize and elaborate on the regulatory mechanisms on A-MN’s oscillatory activity. We further highlighted the similarity between our findings and previous discoveries in other small motor circuits.C. elegans excitatory motor neurons play not only the roles of inducing muscle contraction, but also rhythm-generation, and possibly proprioceptive feedback and coupling, we feel that ‘compression’ better emphasizes the reduction of circuit layers by packing their functions into a single class of neurons.We understand and respect your reservation with the usage of the word ‘compression’. We did not mean to use it in the context of evolution, but wanted to describe a nervous system property that we feel to be more specific than ‘multifunctionality’ . To our understanding, multifunctionality could also be used to describe the same group of neurons or connections to be reconfigured or recruited to generate different motor programs in the same animal. When 12) Title could be shortened and refocused to match the revised Discussion.The title has been shortened.


Spinal motoneurones (Mns) constitute the final output for the execution of motor tasks. In addition to innervating muscles, Mns project excitatory collateral connections to Renshaw cells (RCs) and other Mns, but the latter have received little attention. We show that Mns receive strong synaptic input from other Mns throughout development and into maturity, with fast-type Mns systematically receiving greater recurrent excitation than slow-type Mns. Optical recordings show that activation of Mns in one spinal segment can propagate to adjacent segments even in the presence of intact recurrent inhibition. While it is known that transmission at the neuromuscular junction is purely cholinergic and RCs are excited through both acetylcholine and glutamate receptors, here we show that neurotransmission between Mns is purely glutamatergic, indicating that synaptic transmission systems are differentiated at different postsynaptic targets of Mns. Motoneurones (Mns) are the last elements of the networks that command, coordinate, and actuate the most essential of behaviours: movement. Their activation triggers muscle contractions, and the many diseases affecting Mns cause progressive and fatal paralysis. We show here that Mns themselves form an interconnected network and that activity in one segment of the spinal cord can propagate reciprocally to neighbouring segments, thus constituting a positive feedback loop that can amplify the strength of motor output. Remarkably, while Mns excite muscles through release of the neurotransmitter acetylcholine, our data show that the synapses between Mns operate through glutamate, providing a rare example of differentiation of transmitter systems according to the postsynaptic targets. Motoneurones (Mns) are the ultimate neural targets of effector commands issued from the central nervous system. Their activity is modulated by an intricate network of interneurones  that affEarly anatomical studies  have shoNone of the previous studies provided a comprehensive analysis of the extent of recurrent excitation, and they only illustrated a few recordings of small evoked currents. Furthermore, there are contrasting reports on the type of receptors mediating recurrent excitation, with evidence showing sensitivity to either glutamatergic antagonists , cholineWe performed paired recordings to measure the efficacy of unitary connections in fluorescently labelled Mns innervating gastrocnemius. Simultaneous infrared and confocal imaging was used to identify and patch fluorescent Mns in a dorsal horn–ablated spinal cord . Strychnf]quinoxaline-7-sulfonamide disodium salt (NBQX), to block N-Methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, respectively  and 5 μM dihydro-β-erythroidine (DHβE) to block α7 and αβ receptors, respectively . Group dn = 6, Wilcoxon sign-rank z = −0.53, P = 0.600). The mean latency of the responses was 1.54 ± 0.22 ms, with a jitter of 0.13 ± 0.03 ms.While the data from z = 0.61, P = 0.541). Despite the lack of correlation between the size of the current and the position of the recorded Mn with respect to the stimulated VR, we observed a very broad distribution of amplitudes of rEPSCs, with sizes ranging from 60 to more than 5,000 pA .In the experiments above, the rEPSCs were pharmacologically isolated by blocking fast inhibitory transmission with strychnine and gabazine, in order to avoid potential bias in the current measurements, due to the opening of the large inhibitory conductances associated with the activation of RCs following VR stimulation. However, because the cord might become hyperexcitable following full block of synaptic inhibition, we tested whether propagation of recurrent excitation across segments was preserved even with intact inhibition. In 3 different preparations, we stimulated either L4 or L5 VRs and recorded the rEPSCs from 21 Mns located in the adjacent L5 or L4 segment . Latencies of responses from Mns in neighbouring segments (1.77 ± 0.13 ms) were longer compared to those recorded from within the same segment that was stimulated . The corresponding response jitters were very small both within (0.08 ± 0.03 ms) and across (0.08 ± 0.01 ms) segments, and a comparison between the two showed no significant difference . These results demonstrate that, while the latency of synaptic responses may be greater across segments compared to within segments, they are both mediated by monosynaptic connections between Mns within and across segments.The mean latency (±SEM) of responses of 1.64 ± 0.09 ms was consistent with monosynaptic activation and not significantly different from those from oblique slices . We also performed simultaneous whole-cell recordings of Mns and V2a interneurones, and in n = 9 cells, we could not elicit any synaptic or electrically mediated current from the interneurone .We next assessed whether the magnitude of recurrent excitation was related to the intrinsic properties of postsynaptic Mns. Two types of Mns were identified according to their firing pattern at rheobase in current clamp recordings . The firr|≥0.516, P<0.001, Differences between the 2 cell types are also associated with their passive properties, with delayed-firing Mns showing lower resistances (median 22 MΩ) and higher capacitances (median 237 pF) than their immediate-firing counterparts , both at statistically significant levels . Recurrent excitatory responses were recorded from delayed-firing , purple The presence of strychnine and gabazine during electrophysiological recordings precluded evaluation of whether recurrent excitation could override recurrent inhibition. We therefore conducted calcium imaging experiments in mice selectively expressing GCaMP6s in Mns to evaluate the propagation of recurrent excitation across different segments with recurrent inhibition intact. While the latency of the rEPSCs recorded electrophysiologically was short (1.64–1.77 ms) and exhibited a very low jitter (0.08 ms), a greater variability in the apparent onset of the calcium signals was observed , middle.In control, recurrent excitation evoked spikes in Mns from both L4 and L5 segments, as shown by the running medians and interquartile ranges of the relative fluorescence signal , red thrz = 8.50, P<0.001), and these difference were maintained after block of inhibition . Pooling Mns from both segments, blockade of inhibition consistently increased the signal , whereas a significant reduction in signal was observed following additional application of glutamatergic antagonists . Residual firing was mostly confined to Mns within the stimulated segment through antidromic activation, thus confirming the purely glutamatergic nature of recurrent excitation.Group data from 461 Mns from 7 preparations are shown in hibition , middle hibition , right  would have little effect on the excitability of Mns whose somata are very large, VR stimulation evoked responses usually exceeding 1 nA, indicating extensive convergence of segmental Mn populations.Very few studies have examined, in any detail, recurrent excitation between Mns. In both available electrophysiological studies , 9, the The variation in the magnitude of rEPSCs is associated with Mn classification into delayed and immediate-firing types, with larger responses systematically observed in the delayed-firing, low-resistance, and high-capacitance cells. Our results are therefore consistent with a structural connectivity in which the fast-type larger Mns receive stronger recurrent excitation compared to slow-type smaller cells. This pattern of connectivity suggests that recurrent excitation could play a role in sequential recruitment of fast-type units during motor tasks in which progressively increasing muscular forces are needed. Alternatively, recurrent excitation might represent a closed-loop amplification circuit that reinforces and increases the firing rate preferentially in fast-type Mns and thus rapidly increases muscle contraction strength when required. Distinguishing between these two possibilities would require a full characterization of presynaptic Mns in order to determine whether it is slow or fast Mns that are preferentially connected to fast Mns.In neonatal animals, VR stimulation can induce fictive locomotion  and entrIt is possible, however, that Mns synapse onto other, so far unidentified interneurones that, in turn, could project back to Mns. While the existence of such a disynaptic pathway is possible, it would not account for the large, constant latency responses observed both within and across segments. Indeed, in our electrophysiological recordings during pharmacological blockade of recurrent inhibition, we often observed a late disynaptic component. At present, we cannot ascertain whether the disynaptic current results from premotor interneurons or from orthodromic activation of Mn pools that were not antidromically activated by VR stimulation. In either case, such recruitment implies the existence of a positive-feedback amplifying circuit whose tendency to reverberate may be suppressed by recurrent inhibition.The recurrent excitation characterised in the present study includes predominantly a monosynaptic component, and this is evidenced by three observations. First, the connectivity between Mn pairs must have been monosynaptic. Second, the latency of responses within (1.43 ± 0.09 ms) and between (1.77 ± 0.13 ms) lumbar segments were within the time-scale of neurotransmission through only a single synapse. Finally, the response jitters within (0.08 ± 0.03 ms) and between (0.08 ± 0.01 ms) segments were very small and virtually identical. These observations are only consistent with a monosynaptic connectivity between Mns both within the same segment and across neighbouring segments. While the occurrence of synaptic projections between Mns crossing spinal segments may be regarded as unusual, it is perfectly compatible with the known rostrocaudal distribution of Mn dendritic trees, which may span over 1 millimetre in juvenile mice with little or no change into adulthood .A glutamate receptor–dependent effect on Mn EPSPs evoked by VR stimulation has been reported previously , but it Neurotransmission between Mns is purely glutamatergic, yet following normal maturation, the neuromuscular junction is solely cholinergic  and synaOthers, however, have not detected the presence of VGlut2—or any other vesicular glutamate transporter—in Mn terminals , 22. It All experiments were carried out in accordance with the Animal (Scientific Procedures) Act  and were approved by the UCL Ethical Committee, under project licence number 70/7621. Intramuscular injections were performed under inhaled isofluorane anaesthesia and by a surgical procedure supervised and approved by the Veterinary Surgeon named by the Home Office, UK. Before being euthanized, animals were administered terminal anaesthesia via intraperitoneal injection of a mixture of ketamine and xylazine .Experiments were performed on preparations obtained from male or female mice bred using a C57BL/6J background. For electrophysiological experiments with simultaneous recordings from Mns and RCs, a transgenic strain—in which the EGFP is expressed under the control of the promotor of the neuronal glycine transporter GlyT-2 —was used3, 1 NaH2PO4, 2 CaCl2, 2 MgCl2, and 11 D-glucose (same solution was used for recording). The spinal cord was then glued onto an agar block and affixed to the chamber of a vibrating slicer . We used a slicing solution containing (in mM) 130 K-gluconate, 15 KCl, 0.05 EGTA, 20 HEPES, 25 D-glucose, 3 kynurenic acid, and ph 7.4 with NaOH [Following anaesthesia by intraperitoneal injection of a mixture of ketamine/xylazine , both juvenile and mature mice were decapitated and the spinal cord dissected in normal icecold aCSF containing (in mM) 113 NaCl, 3 KCl, 25 NaHCOith NaOH . For cutFor coronally sliced preparations in which the dorsal horns were ablated, the cord was glued horizontally with the ventral surface facing upwards. A blade was used to transect the cord at the L1–L2 boundary at an angle that allowed visualization of the exact position of the central canal under a dissection microscope. The vibratome blade was then aligned to the central canal, and the ventral portion of the cord was sliced away from the dorsal part. Alignment with the central canal was essential to ensure a consistent dorsoventral level of the ablation across different preparations and to retain the dorsal motor nuclei near the cut surface of the tissue.Identical procedures were used for juvenile (P7–14) and mature (P15–25) animals. For older animals, we routinely cut the first slice within 8 minutes following decapitation. Because spinal cord preparations are extremely sensitive to anoxia, especially prior to slicing, we found that minimizing the time to obtain the first slice consistently resulted in viable preparations with healthy Mns .All recordings from postsynaptic Mns were performed with a Molecular Devices Axopatch 200B amplifier, filtered at 5 kHz and digitized at 50 kHz. Patch pipettes were pulled to resistances in the range of 0.8–2 MΩ when filled with (in mM) 125 K-gluconate, 6 KCl, 10 HEPES, 0.1 EGTA, 2 Mg-ATP, pH 7.3 with KOH, and osmolarity of 290 to 310 mOsm. During voltage clamp recordings, Mns were clamped at −60 mV with series resistances in the range of 2 to 10 MΩ compensated by 60% to 80%.During paired recordings, loose cell–attached stimulation was used to evoke spikes in putative presynaptic Mns using an ELC-03X  amplifier and a 4–5 MΩ pipette filled with normal aCSF . Within VR stimulation was delivered to evoke rEPSCs in Mns using a glass suction electrode whose tip was cut to correspond with the size of the VR . The stiFor measuring the size of the excitatory response in some Mns, for which it was necessary to prevent action potentials, cells were hyperpolarised below their resting membrane potential. Measurements of synaptic current and potentials from these recordings were adjusted to their predicted value at −60 mV, assuming a reversal potential of 0 mV for excitatory conductances. All electrophysiological experiments were performed in the presence of 0.5 μM strychnine and 3 μM gabazine except where stated otherwise. Where indicated, excitatory receptors were blocked using APV, NBQX, MLA, or DHβE.In order to label Mns innervating the ankle flexor gastrocnemius muscle, intramuscular injections were performed 2 to 5 days prior to recording. Inhalant isofluorane was used for the induction and maintenance of anaesthesia. Traction was applied to the lower limb, and an incision was made through the skin and deep fascia overlying the muscle. A Hamilton syringe loaded with a glass needle was used to inject 1 μl of CTB-Alexa-Fluor-555  into the middle of the muscle belly over a period of at least 1 minute. The skin was closed by suture using a buried stitch before cessation of anaesthesia and recovery.Calcium imaging experiments were performed on animals selectively expressing the genetically encoded calcium indicator GCaMP6s in Mns. These mice were generated by crossing mice expressing Cre under the control of choline-acetyltransferase  with animals with the gene expressing GCaMP6 flanked by a flox-Stop cassette (JAX mouse line number 028866 ). Upon rDorsal horn–ablated coronal slice preparations (P9–12) were used for imaging experiments to visualise the dorsal motor nuclei in the L4 and L5 segments containing Mns innervating tibialis anterior, gastrocnemius, and peroneus longus close to the cut surface. A laser-scanning confocal unit  with a diode laser  was used to locate and record calcium signals from Mns reaching a depth of approximately 100 μm from the surface. Fields of 128 × 64 pixels (pixel size 1.38 μm and dwell 7.2 μs) were scanned with a frame interval of 146 ms over different regions throughout the dorsal motor column. Trains of 3 stimulii at 30 Hz were delivered to the VR (L4 or L5) while images were being acquired from at least 1 s before the onset of the first stimulus pulse. For each field, calcium signals were acquired for a total of 35 frames corresponding to approximately 5 s, and the position of each field was recorded.Posthoc analysis was performed to quantify Mn responses. Within each field, single Mns were identified by their fluorescence, and regions of interests were defined by the contour profiles of their somata. The time course of excitation was measured using the change in mean fluoresence following stimulation divided by the baseline average. In some cases, slow drifts in fluorescence was corrected by fitting an exponential to the initial trace before the stimulus. Changes in fluorescence exceeding 2 standard deviations of the baseline noise were measured over a 1 s window following VR stimulation.S1 Data(XLSX)Click here for additional data file.S1 FigDetails of the position of recorded Mns and stimulated VR are shown for 7 preparations. Position and corresponding response in individual Mns are colour coded for each preparation (2 to 9 Mns recorded in each cord). Stimulation artefacts are blanked, and the time of the stimulation is indicated by a coloured segment. Mn, motoneurone; VR, ventral root.(TIF)Click here for additional data file.S2 FigThe position of Mns and stimulated VRs is shown for 3 different preparations in which the rEPSCs were measured in the absence of any antagonists of synaptic inhibition. Responses in 3 to 7 Mns per preparation are shown. Blanked stimulation artefacts are indicated by a coloured line. Mn, motoneurone; rEPSC, recurrent excitatory postsynaptic current; VR, ventral root.(TIF)Click here for additional data file.


Ortho-phenylphenol (OPP) is a fungicide contained in agro-industrial effluents produced by fruit-packaging plants. Within the frame of developing bio-strategies to detoxify these effluents, an OPP-degrading Sphingomonas haloaromaticamans strain was isolated. Proteins/genes with a putative catabolic role and bacterium adaptation mechanisms during OPP degradation were identified via genomic and proteomic analysis. Transcription analysis of all putative catabolic genes established their role in the metabolism of OPP. The formation of key transformation products was verified by chromatographic analysis. Genomic analysis identified two orthologous operons encoding the ortho-cleavage of benzoic acid (BA) (ben/cat). The second ben/cat operon was located in a 92-kb scaffold along with (i) an operon (opp) comprising genes for the transformation of OPP to BA and 2-hydroxypenta-2,4-dienoate (and genes for its transformation) and (ii) an incomplete biphenyl catabolic operon (bph). Proteomics identified 13 up-regulated catabolic proteins when S. haloaromaticamans was growing on OPP and/or BA. Transcription analysis verified the key role of the catabolic operons located in the 92-kb scaffold, and flanked by transposases, on the transformation of OPP by S. haloaromaticamans. A flavin-dependent monoxygenase (OppA1), one of the most up-regulated proteins in the OPP-growing cells, was isolated via heterologous expression and its catabolic activity was verified in vitro. Ortho-phenylphenol (OPP) is used in the post-harvest treatment of fruits to control fungal infestations during storage1. Its application results in the production of large wastewater volumes which require treatment on site2. The development of biological treatment systems based on the specific ability of microorganisms to degrade OPP could be a viable solution for the detoxification of these effluents. In this context, a Sphingomonas haloaromaticamans strain P3 was recently isolated from a soil collected from a wastewater disposal site3. The bacterium is using OPP as a carbon source and showed high potential for application in biodepuration and bioaugmentation strategies. However, the microbial metabolic pathway of the fungicide and the genetic systems driving its degradation by strain P3 remain unknown. Biodegradation is not always synonymous to detoxification; instead it occasionally leads to the formation of metabolic products that are more toxic or persistent than the parent compound4. Therefore, elucidation of the microbial metabolic pathway of OPP is a prerequisite for the downstream exploitation of strain P3 in any environmental application.et al.5 were the first, and the only other study to date, that have studied the microbial degradation of OPP. They isolated a Pseudomonas azelaica strain HPB1 which was able to degrade OPP through the production of 2,3-dihydroxybiphenyl. Jaspers et al.6 identified a gene cluster, hbpCAD, encoding the upper metabolic pathway of OPP which involves the transformation of OPP to 2-hydroxypenta-2,4-dienoateand benzoic acid (BA). The downstream transformation of BA involved a meta-cleavage pathway, although its genetic organization and function was not revealed and the overall network of genes driving the full metabolic pathway of OPP is still not known. HbpA, a flavin-dependent monoxygenase responsible for the initial hydroxylation of OPP by P. azelaica HPB1, was isolated7 and characterized8.To date little is known about the microbial degradation of OPP. Kohler 10, making protein annotation, metabolic pathway prediction and reconstruction feasible12. However, the mere presence of putative catabolic genes in a bacterial genome does not guarantee biodegradability in a given environment13. Proteomic analysis offers a unique dynamic view of the catabolic network of xenobiotic-degrading bacteria within the context of the overall cell response and adaptation to pollutant exposure16. Good knowledge of the physiological response of bacteria during exposure and degradation of organic pollutants is necessary for their future industrial exploitation.Advances in high-throughput sequencing have shed light into the full genetic armoury of xenobiotic-degrading bacteriaS. haloaromaticamans strain P3 during degradation of OPP. To achieve this, a combination of genomics and proteomics coupled with transcription and chromatographic analysis was employed. The total bacterial genome was sequenced, assembled, annotated and used for mapping of the proteome of strain P3 growing on OPP, BA and succinate. Up-regulated enzymes with a putative role in the transformation of OPP were identified and their expression patterns during degradation of OPP and BA was determined by reverse transcription (RT)-q-PCR. The key enzyme involved in the first step of the metabolic pathway, a flavin-dependent monoxygenase, was isolated via heterologous expression, purified and its activity against OPP was verified in vitro. The combinatory use of advanced omic tools is expected to unravel the full genetic network driving microbial degradation of OPP for the first time as part of the wider cellular response of the studied bacterium to fungicide exposure. This knowledge will facilitate the exploitation of S. haloaromaticamans strain P3 in future bioremediation and biodepuration strategies.The aim of the present study was to unravel the genetic mechanisms driving the metabolic pathway of OPP and the overall cellular response of S. haloaromaticamans strain P3 had a size of 4.812.401 bp with a mean GC content of 62%, which falls within the average size and GC content values of known sphingomonad genomes (3.4–5.9 Mbp)17. Variation in the size and the GC content of the genomes of Sphingomonads has been attributed to the presence of plasmids and genomic islands18. A total of 4630 ORFs were predicted. The annotation of the sequenced genome generated 57 large contigs assembled into 13 scaffolds with the main ones being scaffolds 1 (4.5 Mbp), 2 (192 kbp) and 3 (92 kbp).The draft genome of 20. Operons 1 and 2 encoded a BA ortho cleavage pathway (ben/cat operon). Operon 3 comprised genes with a putative role in the upper part of the metabolic pathway of OPP (opp operon) and genes from the lower biphenyl (bph) pathway. Finally operon 4 encoded an incomplete bph pathway (Table 21. The presence of plasmid stabilization proteins (stb) at the 5′ end of operon 3, a near complete Type IV secretion system (virD4 is missing) homologous to the VirB/virD4 secretion system of Agrobacterium tumefaciens23, a conjugal transfer protein (TraG)24 and a chromosome partitioning protein (Spo0J) downstream of operon 425  and then finally to acetyl-CoA and succinyl-CoA via the ortho - cleavage pathway (catABC and pcaDFIJ). A LysR-type transcriptional regulatory protein (benR) was identified at the 5′ end of each operon and was inversely oriented to other genes (Table ben/cat operons showed similar gene organization with the sole differences being: (i) the pcaDF gene organization; in operon 1 pcaF preceded pcaD and (ii) the position of benC, which was located between benB and benD in operon 1, while a putative benC (Ferredoxin–NAD(P)(+) reductase) was localized at the 3′ end of operon 2 , a 2,3 dihydroxy, 1,2-dioxygenase (oppC) and two meta-ring fusion product hydrolases (oppD1 and oppD2) (Table oppD1ACD2), potentially homologous to the hbpCAD gene cluster driving the upper part of the metabolic pathway of OPP in P. azelaica HPB126. The translated products of hbpCAD genes showed 32–45% identities and 51–62% positives to their orthologs OppD1ACD2. Downstream of oppD1ACD2, genes encoding the complete lower biphenyl (bph) pathway were found, including a 2-keto-4-pentanoate hydratase (bphH1), an acetaldehyde dehydrogenase (bphJ) and a 4-hydroxy-2-oxovalerate aldolase (bphI). Denef et al.27 have shown that a similar gene cluster in Burkholderia xenovorans LB400 was responsible for the transformation of 2-hydroxypenta-2,4-dienoate  to acetaldehyde and pyruvate. A transcriptional regulatory gene (oppR) showing high homology to a XylR_N superfamily of σ54-dependent transcriptional regulators was identified upstream of oppD1 . This consists of bphA1A2A3A4 (multi-component biphenyl dioxygenase), bphB (cis-biphenyl dihydrodiol dehydrogenase) and bphD (2-hydroxy-6-oxo-6-(2′-aminophenyl)hexa-2,4-dienoic acid hydrolase), only missing bphC . The latter is known to be responsible for the transformation of biphenyl-2,3-diol to 2-hydroxy-2,4-pentadienoate and benzoate28. The upper bph pathway is ubiquitous in soil bacteria29 and is usually found on transposable elements30. A second bphH2 and a gene encoding a second flavin-dependent monoxygenase (putative oppA2) were both located at the 5′ end of this operon. Two putative transcriptional regulatory genes were identified upstream of the bph genes (bphR1R2).Operon 4 encodes an incomplete upper Sphingomonas and more specifically with proteins from a homologous operon of the HCH-degrading strain Sphingomonas MM131    clustered within the genus S. haloaromaticamans strain P3, a putative metabolic pathway is proposed  and the lower bph pathway (operon 3), respectively. The pathway proposed is similar to the metabolic pathway of OPP by the P. azelaica strain HBP1 with the sole difference of the meta cleavage of BA operated in strain HBP15.Based on the genomic analysis of the sed Fig. , where OS. haloaromaticamans was rapid and concurred with the formation of BA, which peaked at 17 h and rapidly reduced thereafter   a 4-hydroxy-2-oxovalerate aldolase BphI  and chaperonins are essential for the survival of bacteria under stress conditions since they facilitate the correct folding of denaturated proteins in the cytosol36. The mobilization of stress response mechanisms by S. haloaromaticamans cells during degradation of OPP and BA suggests that these compounds are not preferred growth substrates. Previous proteomic analyses with other xenobiotic-degrading bacterial strains have also noted a stimulation of the stress response mechanisms during degradation of BA36, phenanthrene37, and phenylurea herbicides38.Several proteins involved in bacterial stress response were up-regulated in OPP- and BA-grown cells  and proteins involved in membrane permeability and stability  was detected in the proteome of OPP-grown cells H nitroreductase, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase) and in the synthesis of biomolecules  were up-regulated in OPP- and/or BA-grown cells  showed similar patterns with significantly higher expression (p < 0.05) in the presence of OPP compared to BA and succinate , showed a significantly higher expression in the presence of OPP compared to BA- and succinate-grown cells , suggesting an auxiliary role in the degradation of OPP. This is in agreement with the low substrate specificity of biphenyl dioxygenases45 and the structural resemblance of OPP and biphenyl, whose transformation converges on the same intermediates acting as weak effectors of the bph pathway. In contrast to the other genes of the operon, bphD showed expression levels equivalent to oppD1 from operon 3  showed significantly higher expression when grown on OPP  in operon 3, further verifying the prime role of operon 3 in the transformation of OPP and its intermediates like 2-hydroxypenta-2,4-dienoate.Genes lls Fig. . The bphben/cat operon showed uniform expression patterns. Genes from both operons showed significantly higher expression in BA- (p < 0.05) compared to OPP- and succinate-grown cells  in the expression levels only of the operon 2 genes. When the expression level of orthologous genes of the two ben/cat operons were compared, a significantly higher expression of all genes from operon 2 was evident, suggesting that S. haloaromaticamans activates operon 2 for the metabolism of BA formed by OPP transformation. Considering the localization of operon 1 in the bacterial chromosome, it is tempting to assume that this operon is utilized by the bacterium for the consumption of biogenic aromatic organic acids, like substituted benzoic and cinnamic acids with methoxy or hydroxy substituents46, which thrive in the soil environment. Ben/cat operons are ubiquitous in soil bacteria playing a central role in the degradation of biogenic aromatic compounds encountered in soil47. Alternatively, ben/cat operon 1 might be regulated differently depending on the concentration of BA or the growth stage of the bacterium. Denef et al.48 showed that the biphenyl-degrading strain Burkholderia xenovorans LB400 could concurrently utilize two or three pathways to transform BA depending on the initial substrate (BA or biphenyl) and its growth stage.All genes from the same lls Fig. . Whereasopp and upper bph) and two central catabolic pathways (ben/cat and lower bph) in S. haloaromaticamans is contrary to the modular construction of catabolic pathways found in most xenobiotic-degrading bacteria, where several peripheral metabolic pathways are funnelling their products into a single central catabolic pathway49. The phylogenetic classification of the genes of the two ben/cat operons and the flanking of operon 2 by transposable elements supports its lateral acquisition from a phylogenetically close bacterium through horizontal gene transfer, rather than through duplication and gradual evolution of operon 1, a strategy commonly utilized by other oligotrophs (i.e. Arthrobacters)50. Sphingomonads are known to utilize horizontal gene transfer as a major mechanism to expand or optimize their catabolic capacities21. We suggest that S. haloaromaticamans has evolved its capacity to metabolize OPP by gradual acquisition and assemblage of operons 2 and 3 from taxonomically close and distant bacteria, respectively. While the acquisition of operon 3 by S. haloaromaticamans was a key step towards the development of its capacity to transform OPP, the acquisition of operon 2 is most probably an optimization step towards a more efficient metabolism of BA.The presence of two peripheral  indicating a transcriptional repressor activity. Vasely et al.51 reported a repressor activity of catR on the catABC locus of a Rhodococcus erythropolis strain, whereas other studies have shown that catR acts as an activator of the ortho-cleavage pathway52. The presence of a single regulatory protein BenR for both segments of the pathway (ben and cat) has been reported previously in xenobiotics-degrading sphingomonads31 and it was identified as an effective transcription regulation mechanism induced by both BA and cis, cis-muconate53.p < 0.05) in the presence of OPP   during degradation of OPP suggesting a transcriptional activator regulatory role  as responsible for the initial hydroxylation of OPP. The flavin-dependent monoxygenase encoded by this gene was isolated via heterologous expression in E. coli and purified , and a dichlorophenol hydroxylase from another Sphingomonas isolate  (MSMN + CA)3. The medium was amended with filter sterilized aqueous solutions of OPP (200 mg L−1), BA (200 mg L−1) and succinate (2000 mg L−1). Bacterial growth was determined by optical density at 600 nm (OD600). Analytical standards of OPP (99.9%), BA (99.5%), 2,3-dihydroxybiphenyl (≥98%), catechol (≥99%) were purchased from Sigma-Aldrich  and succinate (99%) from PanReac-AppliChem .The S. haloaromaticamans with the Purelink Genomic DNA Mini kit  and quantified by Qubit . Sequencing was performed by Illumina MiSeq with a 2 × 300 bp paired-end (insert ~550 bp) and a 2 × 300 bp mate-pair (insert ~3000 bp) runs. Genome assembly was performed with Allpaths-LG v5096058 using the default parameters. Genome completeness and purity was checked with the CheckM v0.9.6 software suite59 and annotation of the resulting contigs was performed with Prokka v1.1060 as described in details in the Supplementary Information , + BA (62 mg L−1) or + succinate (100 mg L−1) were inoculated with S. haloaromaticamans as described above, aiming to equivalent carbon concentration in all treatments (42.5 mg C L−1). Duplicate non inoculated MSMN + CA + OPP or MSMN + CA + BA were also prepared. All samples were incubated in an orbital shaker at180 rpm and 26 °C. The degradation of OPP and BA and the growth of S. haloaromaticamans was determined at regular intervals by HPLC and OD600 measurements, respectively. When bacterial growth in all treatments reached the mid-log phase (OD600 = 0.15–0.16) bacterial cells were harvested by centrifugation  and used for protein extraction. Bacterial pellet was kept on ice and re-suspended in cold buffer A . Upon addition of 0.1% TRITON and 0.5 mM PMSF the cells were ultrasonicated on ice three times for 15 sec. Cell debris were removed by centrifugation .Triplicate cultures of MSMN + CA + OPP  and then by SDS–PAGE using 12.5% Tris-HCl polyacrylamide gels (Bio-Rad) following standard procedures. For each treatment three biological replicates were run in parallel and silver stained. 2-DΕ gels were scanned with Bio-Rad GS-800 Calibrated Densitometer equipped with PDQuest Advanced 2-DΕ Gel Analysis software  as previously described64. Data were analyzed by one-way ANOVA (P ≤ 0.05) and means were compared using Student’s t-test (significance level 95%). The statistical significant differences were further combined by the quantitative 2-fold change of spot volume. Spots showing values in the ratios OPP/Succinate and BA/Succinate (volume intensity) lower than 0.5 or higher than 2 were excised from the 2D-PAGE gels and digested with trypsin (more details are available in the Supplementary Information section SI 1.3.).For 2D-PAGE separation crude protein extracts were further clarified, concentrated and protein pellet was solubilized at the desired volume of rehydration buffer as described beforeS. haloaromaticamans P3 & Uniprot-Trembl databases. The mass error tolerance on the Mascot server was set to 25 ppm, methionine oxidation was considered as a variable modification and cystein carbamido methylation was considered as a fixed modification. Proteins not identified by MALDI-TOF analysis were reanalyzed by HPLC-tandem MS/MS (Thermo Scientific) as described in the Supplementary Information . The protein identification was carried out by peptide mass fingerprinting on a locally installed Mascot-Server v 2.0 against the genome of Bacterial pellet collected at 4, 10, 12 and 27 h during the proteomics experiment was stored at −80 °C and used for transcription analysis of putative catabolic genes. RNA was extracted with the Nucleospin RNA II kit . In most cases a DNAse treatment step  was essential to remove DNA residues from extracted RNA. DNA-free RNA was then reverse transcribed to obtain cDNA  using random hexamers .S. haloaromaticamans were manually designed with the program PrimerSelect™  based on the genomic analysis of the studied strain  and by PCR, using total DNA of strain P3, and sequencing of the PCR product obtained. Primers for the amplification of the gyrB gene of S. haloaromaticamans  were also designed based on the genome of the strain P3 . Statistical analysis was performed with the SPSS Statistics (IBM Corp. Version 21.0.) software.Primers for the amplification of all putative catabolic genes of oppA1 (oppAf 5′ACTCATGGATCCATGACTTCAGCAGTTCAAAAACCG3′ and oppAr 5′ACTCATCTCGAGTTAAGAAGCCGGCGTGAATTTTG3′). Underlined nucleotides indicate restriction sites for enzymes BamHI and XhoI to facilitate ligation into the plasmid vector pGEX-6P-1 . Amplification of the putative oppA was performed in 50 μl reactions containing 1X Polymerase Buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.5 μΜ of each primer, 1 U of Thermo Scientific Phusion High-Fidelity DNA Polymerase and 1 μl of total bacterial DNA. Amplification conditions were 98 °C for 30 sec, 30 cycles of 98 °C for 10 sec, 67 °C for 30 sec and 72 °C for 1 min, and final extension at 72 °C for 10 min. The product was purified, digested with BamHI and XhoI and ligated into the pGEX vector digested with the same enzymes. Plasmids were transformed into Escherichia coli BL21(DH3) cells.Primers were designed to amplify the full length sequence of E. coli transformed cell cultures were grown in LB + ampicillin (100 μg mL−1) in an orbital shaker (200 rpm) at 37 °C to an OD600 of 0.6. The recombinant enzyme was induced with IPTG (0.025 mM) and cells were harvested, 16 h after induction, by centrifugation at 8000 rpm at 4 °C for 7 min. Cells were re-suspended in Buffer A and lysed with ultrasonication (6 × 10 sec). The samples were centrifuged for 15 min at 14500 rcf at 4 °C. The soluble proteins in the supernatant were collected and purified by passage through pre-equilibrated Protino Glutathione Agarose 4B . The suspension was mixed gently at 4 °C for 2 h. The gel was centrifuged for 5 min at 500 xg and the supernatant was discarded. The gel was resuspended in 100 μl of buffer A, transferred to an appropriate chromatography column (Micro Bio-Spin™ Chromatography Columns) and washed successively with the solutions buffer A, buffer A + Triton (0.2%), buffer A and buffer A + DTT (10 mM). Then, the column outlet was closed with cap and the gel was incubated overnight at 4 °C with buffer A + 3 C protease (55 μg). After the incubation the eluate was collected and the purified protein was run with SDS-PAGE66. The gel was stained with Coomassie Brilliant Blue R-250 and destained with a solution of 30% methanol and 10% acetic acid in deionized water. The activity of the purified enzyme was tested in 1-ml reactions composed of 0.25 mM of OPP, 1 mM of NADH, 20 mM phosphate buffer (pH 7.5) and three protein amounts, 9.6, 32 and 96 μg. Triplicate reactions per protein level were prepared, while triplicate controls without protein were also included. The degradation of OPP was determined by HPLC-UV at 0, 9 and 21 h.Eight hundred ml of −1) cultures were inoculated with S. haloaromaticamans. Triplicate non-inoculated controls were also included as abiotic controls. Immediately after inoculation and at hourly intervals thereafter the degradation of OPP and the formation of the putative metabolites 2,3-dihydroxybiphenyl, BA and catechol were determined by HPLC-UV. All chemicals were extracted from 0.5-mL aliquots and analysed as described before3.Triplicate MSMN + CA + OPP (50 mg LThe assembled genome of the strain P3 has been deposited at DDBJ/ENA/GenBank under the accession number MIPT00000000. All data generated or analysed during this study are included in this published article and its Supplementary Information files.Supplementary InformationSupplementary Table S1Supplementary Table S2