TC-specific genetic material profiles, ion channels, cytoskeletal elements, Tps dynamics, etc., (ii) impaired TCs-mediated multicellular signalling, such as homeostasis/angiogenesis, tissue repair/regeneration, neurotransmission, (iii) derangement of 3-D network and impaired mechanical support for TCs-mediated multicellular signals within the stromal compartment, consequently induced interstitial fibrosis, (iv) involvement in local inflammatory process/ immunoregulation and possibly immune-mediated early pregnancy failure.Acute salpingitis (AS) is an inflammatory disease which causes severe damage to a subset of classically described cells lining in oviduct wall and contributes to interstitial fibrosis and fertility problems. Telocytes (TCs), a newly discovered peculiar type of stromal cells, have been identified in many organs, including oviduct, with proposed multiple potential bio-functions. However, with recent increasing reports regarding TCs alterations in disease-affected tissues, there is still lack of evidence about TCs involvement in AS-affected oviduct tissues and potential pathophysiological roles. We presently identified normal TCs by their characteristic ultrastructural features and immunophenotype. However, in AS-affected oviduct tissues, TCs displayed multiple ultrastructural damage both in cellular body and prolongations, with obvious loss of TCs and development of tissue fibrosis. Furthermore, TCs lose their interstitial 3-D network connected by homocellular or heterocellular junctions between TCs and adjacent cells. And especially, TCs connected to the activated immunocytes  and affected local immune state (repression or activation). Meanwhile, massive neutrophils infiltration and overproduced Inducible Nitric Oxide Synthase (iNOS), COX-2, suggested mechanism of inflammatory-induced TCs damage. Consequently, TCs damage might contribute to AS-induced structural and reproductive functional abnormalities of oviduct, probably  Pelvic inflammatory disease (PID) is an inflammatory disease which frequently causes fertility problems among women of reproductive age et al. etc. (www.telocytes.com) Telocytes (TCs) were recently identified as a peculiar type of stromal cells by Popescu On the basis of our previous observations Three-month-old virgin, female, Sprague–Dawley rats (200–250 g) were provided by the Laboratory Animal Center of Soochow University . All rats were maintained in specific pathogen free conditions under environmentally controlled temperature of 21 ± 1°C, and were acclimatized to 12 hrs day/night cycle for at least 10 days before being used for experiments. The study protocol was approved by the Institutional Ethics Review Board of Soochow University.Escherichia coli  was used for induction of AS. Single colony of a pure culture of E. coli was transferred and incubated overnight for 18 hrs at 37°C, aerobically in the dark. For stock cultures, beef extract decoction agar slants were inoculated and similarly incubated, until an optical density equivalent to 109 cfu/ml were acquired via lower midline abdominal incision, both oviducts were identified and bacteria liquid  were injected into its lumens in AS group. Applying the same amount of sterile saline instead of bacterial liquid served as the sham controls. Then, the abdominal wall was closed and maintained in the same conditions.Thirty rats were randomly divided equally into AS and sham control group. Rat model of AS-affected oviduct was established 3) were fixed in 10% formalin and embedded in paraffin. Serial sections (5 μm) were subjected to haematoxylin and eosin, immunohistochemistry (IHC) and immuofluorescence experiments. The rest of fresh oviduct tissues (1 mm3) were used for ultrastructural observation of TCs by transmission electron microscope (TEM).At the seventh day after surgery, when rat model of AS were successfully developed To evaluate neutrophils infiltration, sections were processed for routine IHC using rabbit anti-rat polyclonal CD177 . Omission of the primary antibody served as the negative control.iNOS and COX-2 were quantitatively measured by single-labelled immunofluorescence. Briefly, sections were incubated with primary anti-rat antibodies: rabbit polyclonal anti-iNOS ; mouse polyclonal anti-COX-2 . Then FITC-goat anti-rabbit/mouse IgG  was added. Finally, the sections were mounted with antifade medium . Immunofluorescence intensity was quantitatively measured by laser confocal scanning microscope . Images were converted to greyscale and pixel values of positive-stained areas were used for staining intensity. Total staining intensity per selected area was calculated by multiplying the number of pixels/area with the area mean intensity. At least 500 cells obtained from three separate areas/microscopical fields for iNOS  were analysed for each experimental condition using Image-Pro plus software  versus CD34, vimentin versus c-kit): mouse anti-rat monoclonal vimentin , rabbit anti-rat polyclonal CD34 , rabbit anti-rat polyclonal c-kit . Then FITC-goat antimouse IgG for vimentin , CY3-goat anti-rabbit IgG for CD34 , TRITC-goat anti-rabbit IgG for c-kit  were added . Finally, counterstained with DAPI  and mounted with antifade medium . Sections were observed under fluorescence microscope . Telocytes was the only kind of cell which were double-positive for vimentin and CD34, with typical TCs morphology and intact nuclei Generally, double-labelled immunofluorescence using CD34 in combination with PDGFR alpha or beta, c-kit and vimentin was the available choice for TCs immunodiagnostics 3) were processed for routine epon-embedding procedures Tissue fragments . P < 0.05 was considered to be statistically significant.Values of the number of TCs, immunofluorescence intensity of iNOS and COX-2 were presented as mean ± SD, and analysed with Student’s i) smooth muscle cells (SMCs) and capillaries swelling, inflammatory congestion and exudation, interstitial oedema and infiltration of neutrophils  extensive infiltration of numerous neutrophils  in AS group demonstrated dilated lumen, wall congestion and peritubal adhesions. On microscopic examination, specimens from AS group presented with acute inflammation reaction as follows  exhils Fig. and D, ahils Fig.. Howeverhils Fig., with nohils Fig..P = 0.000; Fig.P = 0.000; Fig.By using single-labelled immunofluorescence, the expression of iNOS and COX-2 were visualized. Quantitative analyse under laser confocal scanning microscope showed that in AS group, the expression level of iNOS and COX-2 were significantly higher than that in the sham group: iNOS (P = 0.000), which demonstrated rich double immunostaining for CD34/vimentin  were detected, overlapped with vimentin positive immunofluorescence (green) in the merged images. Meanwhile, there were no cells demonstrated co-expression of vimentin and c-kit (images not shown). Moreover, CD34/vimentin double-positive cells demonstrated a small cell body , with two or more extremely long/thin prolongations surrounding the capillaries, as well as double immunofluorescence in its full length Fig., thus contin Fig..The existence of a novel and peculiar interstitial cell type named TCs in oviduct tissues was observed under TEM. Telocytes had a slender piriform/spindle/triangular-shaped cellular body, with extremely long/thin and moniliform Tps, frequently two or more per TCs Figs and 5A–DTelocytes were observed frequently enwrapping whole capillaries with their long Tps, or scattered in SMCs bundles in mucosa and muscular layer of oviduct Figs and 5D. In AS-affected oviduct tissues, TCs was obviously decreased or lost, severely damaged/degenerated with multiple ultrastructural abnormalities, such as loss of organelles, numerous swollen nucleus, mitochondria and rough endoplasmic reticulum dilatation, cytoplasmic vacuolization, discontinue or dissolution of Tps, swollen or loss of intercellular junctions Fig.–D. The oAcute salpingitis was the most important component of PID spectrum e.g. fibroblasts, mesenchymal SCs, lymphocytes or endothelial cells, with specific roles in cell signalling, tissue or fibrosis remodelling, angiogenesis, physical and/or chemical sensors, not only by their specific ultrastructural configuration + channels are present in TCs and might have significant implications in myocardial physiopathology To date, a number of studies on TCs have yielded constant results regarding the identification, distribution, ultrastructure and immunophenotype in various normal cavitary and non-cavitary mammal tissues and organs, including heart e.g. capillaries, SMCs, activated immunocytes and SCs, TCs formed a complex 3-D extracellular network within the stromal compartment. Meanwhile, TCs occupied a critical position in such specific 3-D structure, indicated roles of mechanical support for intercellular signalling, possibly through chemical (short distance) interaction and physical (long distance) interaction Presently, normal oviduct TCs was identified and confirmed the literatures description e.g. Tps length, podomer thickness and podoms’ diameters under TEM, as previously reported in human normal myometrium via the follows, although none of them has yet been proven definitively.Currently, following our recent study on TCs in endometriosis-affected oviduct tissue, we herein reported for the first time that TCs displayed inflammatory-induced extensive ultrastructure damage, with obvious cell loss, decrease or almost complete absence in AS-affected oviduct tissues. Unfortunately, owing to degeneration, swollen or enlarged, discontinue or dissolution of TCs and Tps, we could not perform comparative morphometric analysis between AS-affected and -unaffected oviduct tissues, e.g. TC-specific genome, proteomic profiles and microRNA signature, ion channels, neurotransmitter, cytoskeletal elements, etc. Then substances and energy insufficiency will decrease basic activities of TCs, such as electrophysiology, dynamics of Tps in terms of adherence, spreading/extension and ramification. Second, swollen or loss of intercellular contacts between TCs and neighbouring cells , will impair multicellular signalling and proposed bio-functions, regarding homeostasis/angiogenesis, tissue repair/regeneration, neurotransmission/muscular contraction, immunoregulation.First, ultrastructure damage or loss of multiple subcellular organelles within cellular body and Tps, will inevitably influenced metabolic pathways, including substances synthesis and energy metabolism, presumably, etc.), for multicellular signals integrating and coordination, and for the correct organization of extracellular matrix components. Derangement of 3-D network will alter the spatial relationships of Tps with neighbouring cells; induce disintegrating of different structural components of interstitium, until finally cause interstitial fibrosis. Interestingly, it was reported that, TCs loss might precede the onset of fibrotic remodelling of the intestinal wall, rather than being merely a consequence of the fibrotic process Third, TCs and Tps damage will change its ultrastructural configuration and the strategic extracellular 3-D architecture, which was structural basis or mechanical support for the guidance of cell migration  release of various cytotoxic cytokines and enzymes into intratubal fluids, which will potentially lead to sperm phagocytosis, impaired fertilization, defective early embryo growth, abnormal transportation and implantation, finally cause immune-mediated failure of earlier pregnancy, including TFI and TEP.via above-mentioned mechanisms. In addition, similar to the role of local immunoregulation through heterocellular junctions between TCs the activated immunocytes  in AS, synapse between Tps and activated mast cells, also indicated TCs involvement in local inflammatory response and possibly associated tubal factor fertility problems in endometriosis-affected oviduct tissues Even more importantly, the findings of inflammation-induced TCs damage in infection based inflammatory AS was consistent with, and further strengthen the idea of a broad involvement of TCs in our previously reported endometriosis-affected oviduct tissue, which was an aseptic inflammatory, ischaemic, hyperoestrogenic condition i) substance and energy insufficiency with deactivation of basic cell bio-functions, e.g. TC-specific genetic material profiles, ion channels, cytoskeletal elements, Tps dynamics, etc., (ii) impaired TCs-mediated multicellular signalling, such as homeostasis/angiogenesis, tissue repair/regeneration, neurotransmission, (iii) derangement of 3-D network and impaired mechanical support for TCs-mediated multicellular signals within the stromal compartment, consequently induced interstitial fibrosis, (iv) involvement in local inflammatory process/ immunoregulation and possibly immune-mediated early pregnancy failure. Nevertheless, it will be of great importance to investigate the underlying molecular and cellular mechanisms targeting oviduct TCs, and TCs-mediated pathological alterations, such as differentiation/activation of fibroblasts and immunocytes. Meanwhile, further functional studies are necessary to better identify the pathophysiological role of TCs in the setting of different oviduct disease.In conclusion, this is the first report on inflammatory-induced ultrastructural alterations of TCs in AS-affected oviduct tissues, presented with obvious damage and loss of TCs and its intercellular junctions accompanying the fibrotic remodelling of oviduct wall. And especially, TCs was found to participate in local immunoregulation. On the basis of these observations, TCs damage might further cause: (


Pulmonary mucormycosis is commonly encountered in patients with diabetic ketoacidosis, hematologic malignancies, neutropenia, organ or hematopoietic stem cell transplantation, and malignancy, but it rarely occurs in high-risk patients with systemic lupus erythematosus (SLE). We present the case of a 40-year-old SLE female with fulminant pneumonia after remission of nephritis treated with rituximab, who developed severe pulmonary mucormycosis that led to her rapid death from acute respiratory failure and acute respiratory distress syndrome. Pulmonary mucormycosis has a high mortality rate. However, with early diagnosis and antifungal therapy with lipid formulation-liposomal amphotericin B and surgical removal of the infected area, the outcome can be improved. Rhizopus 47% and Mucor 18%) are of the order Mucorales. These include Rhizopus, Mucor, Cunninghamella, Apophysomyces, Absidia, Saksenaea, Rhizomucor, Cokeromyces, and Syncephalastrum species. Infections caused by both mucormycosis and entomophthoramycosis were previously called zygomycosis. The class Zygomycetes has disappeared from current taxonomy. The infections caused by the order Mucorales are now termed mucormycosis [The most common invasive fatal opportunistic diseases , diabetic ketoacidosis, metabolic acidosis, persistent neutropenia, high-dose glucocorticoid therapy, trauma or burns, and iron overload due to chelation therapy with deferoxamine in patients on dialysis or chronically transfusion dependent . InvasivThis paper describes a patient receiving rituximab for SLE nephritis after remission that presented with severe pneumonia which was complicated by acute respiratory distress syndrome because of invasive mucormycosis.μL , and platelet counts of 145,000 cells/μL. Blood biochemistry revealed lactate dehydrogenase (LDH) 1097 U/L; procalcitonin (PCT) 0.26 ng/mL; C-reactive protein (CRP) 11.1 mg/dL . On hospital day 7, a lymphocytes cell laboratory testing through flow cytometry revealed white cell counts of 4,090 cells/μL , absolute lymphocyte count 353 cells/μL (9%), absolute CD19 B lymphocyte 64 cells/μL (18.1%), absolute CD4 T-cell count 177 cells/μL, and absolute CD8 T-cell count 127 cells/μL. A chest X-ray after admission revealed the presence of bilateral infiltration and poorly defined nodular, cavitary opacities in both lower lungs , a heart rate of 140/min, and respirations at 24/min. Blood pressure was 142/89 mm Hg and oxygen saturation on room air was 90%. A complete blood count revealed hemoglobin levels of 11.4 g/dL, white cell counts of 2,010 cells/ Figures  and a cl Figures . Microbi Figures . By now There has been a marked increase in the incidence of opportunistic fungal infections worldwide and an even more pronounced increase with mucormycosis , 8. The Long-term corticosteroid therapy enhances a patient's susceptibility to mucormycosis by causing defects in macrophages and neutrophils , 12. The Rhizopus species after her death.At first, she suffered from dyspnea and fever intermittently for 7 days. A chest X-ray revealed both cavitary lower lungs and opacity infiltration. Hence, the tentative diagnosis was SLE relapse and a pulmonary infection. The high level of CRP was probably indicative of a bacterial or fungal infection. She was initially treated with a broad-spectrum antimicrobial agent and preemptive antifungal agent such as fluconazole and micafungin. The clinical course of pneumonia was so fulminant that despite using ECMO in the ICU the patient still died. The sputum culture showedPulmonary mucormycosis develops after inhalation of fungal sporangiospores, by spreading from hematogenous or lymphatic system to the lungs or angioinvasion causing necrosis and infarction of the affected tissues . PulmonaDirect examination of sputum, paranasal sinus secretions, or BAL fluid is frequently nondiagnostic, but isolation of Mucorales organisms in such specimens from a susceptible host with pneumonia or rhinocerebral infection should be considered a strong indicator of mucormycosis . Direct This patient developed ARDS and was quickly treated with antimicrobials. We believe that a delay in appropriate antifungal treatment probably contributed to the poor outcome. It is important to distinguish mucormycosis from aspergillosis because the treatments can differ and because early appropriate antifungal therapy of mucormycosis may improve outcomes , 12. EffIn summary, mucormycosis occurs most frequently in patients with diabetes mellitus, neutropenia coupled with hematological diseases, and organ transplants and patients receiving immunosuppressive medication . Pulmona


Objective: Memorial Delirium Assessment Scale (MDAS) assesses severity of delirium. However, whether the MDAS can be used in a Chinese population is unknown. Moreover, the optimal postoperative MDAS cutoff point for describing postoperative delirium in Chinese remains largely to be determined. We therefore performed a pilot study to validate MDAS in the Chinese language and to determine the optimal postoperative MDAS cutoff point for delirium.Methods: Eighty-two patients , who had hip surgery under general anesthesia, were enrolled. The Confusion Assessment Method (CAM) and Mini-Mental State Examination (MMSE) were administered to the patients before surgery. The CAM and MDAS were performed on the patients on the first, second and fourth postoperative days. The reliability and validity of the MDAS were determined. A receiver operating characteristic (ROC) curve was used to determine the optimal Chinese version MDAS cutoff point for the identification of delirium.Results: The Chinese version of the MDAS had satisfactory internal consistency (α = 0.910). ROC analysis obtained an average optimal MDAS cutoff point of 7.5 in describing the CAM-defined postoperative delirium, with an area under the ROC of 0.990 .Conclusions: The Chinese version of the MDAS had good reliability and validity. The patients whose postoperative Chinese version MDAS cutoff point score was 7.5 would likely have postoperative delirium. These results have established a system for a larger scale study in the future. Delirium is a disturbance of consciousness with an acute onset and a fluctuating nature, that is accompanied by changes in cognition or perceptual disturbances which are not attributable to pre-existing psychiatric disorders or substance-induced states  age 65 years or older; (2) patients who had hip replacements or open reductions with internal fixation (ORIF) under general anesthesia for the repair of hip fractures. Patients were excluded from participation if they had: (1) cognitive impairment at enrollment (MMSE scores less than 18); and/or (2) pre-existing delirium, cerebrovascular disorders or mental disorders , diagnosed by using the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) , medical information , and evaluation of cognitive function using the Mini-Mental State Examination (MMSE). The CAM was also performed on the participants one day before the surgery.All of the participants had hip replacements or open reductions with internal fixation under general anesthesia for the repair of hip fractures. The participants had standardized perioperative care, including preoperative medication, general anesthesia, and postoperative pain control. The participants were given midazolam  as preoperative medication. The general anesthesia was induced by intravenous administration of propofol (95.56 ± 42.51 mg), sufentanil (14.45 ± 6.51 µg), and cisatracurium (10.52 ± 4.51 mg). The general anesthesia was maintained by using propofol (295.66 ± 121.14 mg), remifentanil (0.89 ± 0.28 mg), sevoflurane (21.54 ± 6.78 ml), and cisatracurium (7.52 ± 3.45 mg). The postoperative pain control included a standard postoperative pain management, e.g., sufentanil and butorphanol patient-controlled analgesia . There were no major complications among the participants during the immediate postoperative period.The assessment of delirium was performed after surgery once per day between 8:00 am and 2:00 pm. Patient charts were not reviewed for episodes of delirium, which could have occurred outside the time of assessment. The prevalence of postoperative delirium was assessed by a psychiatrist, (Yujie Wu), according to the CAM diagnostic algorithm. The severity of delirium was determined with the MDAS by another psychiatrist, (Zhongyong Shi), who was blinded to the results of the CAM. The psychiatrists who performed the delirium assessments in the current study had good training and went through quality control procedures. In the current study, the CAM and MDAS were conducted on the first (day 1), second (day 2) and fourth (day 4) day after the surgery by these psychiatrists, because postoperative delirium occurs most often on postoperative day 1 and 2. We performed the CAM and MDAS on postoperative day 4 to detect late-occurring postoperative delirium cases.The CAM algorithm consists of four clinical criteria: (1) acute onset and fluctuating course; (2) inattention; (3) disorganized thinking; and (4) altered level of consciousness. For delirium to be defined, both the first and the second criteria have to be present, plus either: the third or the fourth criteria present, or both the third and forth criteria present together  awareness; (2) orientation; (3) short-term memory; (4) digit span; (5) attention capacity; (6) organizational thinking; (7) perceptual disturbance; (8) delusions; (9) psychomotor activity; and (10) sleep-wake cycle  with P < 0.05 as the significance level.Participants’ characteristics, including age, height, weight, education, length of anesthesia, length of operation, estimation of blood loss and MDAS and MMSE scores, were presented as means ± standard deviation (SD). Continuous parameters were compared with the Analysis of Variance (ANOVA) or Student Reliability was determined by using Cronbach’s alpha coefficient. The Cronbach’s alpha coefficient was calculated to assess internal consistency between MDAS items (inter-item reliability).t-test, comparing the MDAS scores  between patients with or without delirium, as determined by the CAM. The hypothesis was that higher MDAS scores would be associated with the presence of CAM-defined delirium.Concurrent validity of the MDAS was evaluated by Student A receiver operating characteristic (ROC) curve was used to determine the optimal MDAS cutoff point for description of CAM-defined postoperative delirium. The total area under the curve (AUC), its 95 % confidence interval (CI), the total accuracy and the Kappa value, were all used for this determination. The optimal MDAS score was calculated as: (maximum of [sensitivity + specificity − 1]).N = 43) or ORIF (N = 38) surgeries. Twenty-one of the 82 participants (25.6%) developed postoperative delirium either on day 1, day 2 or day 4. The prevalence of delirium in this patient population on day 1, day 2 and day 4 were 17 (20.7%), 17 (20.7%) and 13 (15.8%), respectively.One hundred and thirty patients were initially screened, and a total of 82 patients were included in the final data analysis  showed strong (r ≥ 0.7) or moderate (0.5 ≤ r < 0.7) correlation with the MDAS total score. Item 10 had a weak correlation with the total MDAS score (r < 0.5) .Moreover, the participants who developed postoperative delirium had a higher MDAS score  than the participants who did not develop postoperative delirium: 11.44 ± 4.81 vs. 3.14 ± 1.81 (P < 0.001) . The positive and negative predictive values of this MDAS score (7.5) were 0.918 and 0.990, respectively  patients   Table . These fThere were several limitations in the current study. First, patients were assessed for delirium only on postoperative days 1, 2 and 4, but not on day 3 or on later days after the surgery . However, most cases of postoperative delirium occur in the first 2 days after surgery. In addition, we only included participants who had hip replacements or open reductions with internal fixation under general anesthesia for the repair of hip fractures in the current study. It is possible that patients who have different types of surgeries  may have different optimal MDAS scores for the purpose of identifying postoperative delirium. Future studies may need to include patients undergoing other kinds of surgeries.In conclusion, the results from the current study show that the MDAS in the Chinese language could be an effective and reliable method for determining the severity of delirium symptoms in older Chinese adults. Moreover, a cutoff score of 7.5 was discovered to have a very strong agreement with the CAM algorithm and can therefore be used to diagnose delirium. The findings from this pilot study have established a system and have provided preliminary data for future, larger-scale research with a Chinese population on postoperative delirium determined by using both the CAM and the MDAS. With the establishment of this current system, we should be able to use the Chinese version of the MDAS in the future to assess the severity of postoperative delirium in different Chinese populations.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila melanogaster that can elicit stereotyped leg movements that groom the antennae. Mechanosensory chordotonal neurons detect displacements of the antennae and excite three different classes of functionally connected interneurons, which include two classes of brain interneurons and different parallel descending neurons. This multilayered circuit is organized such that neurons within each layer are sufficient to specifically elicit antennal grooming. However, we find differences in the durations of antennal grooming elicited by neurons in the different layers, suggesting that the circuit is organized to both command antennal grooming and control its duration. As similar features underlie stimulus-induced movements in other animals, we infer the possibility of a common circuit organization for movement control that can be dissected in Drosophila.Animals perform many stereotyped movements, but how nervous systems are organized for controlling specific movements remains unclear. Here we use anatomical, optogenetic, behavioral, and physiological techniques to identify a circuit in DOI:http://dx.doi.org/10.7554/eLife.08758.001 Many movements that animals perform regularly—including walking and grooming—consist of stereotyped sequences of muscle contractions. For example, a dog may scratch its side in response to a fleabite or because it is itchy. But how does the nervous system trigger such specific movements from among the repertoire of different movements that the animal could perform? It also remains unclear how such movements can be produced in a reliable, yet flexible manner.Drosophila uses to groom its antennae. Such grooming movements are stereotyped yet have certain degrees of flexibility, which makes them ideal to study the neural circuits that underlie specific movements. Grooming further lends itself to this kind of investigation because it can be triggered by irritating the surface of the fly's body. There is also an extensive genetic toolkit that can be used to manipulate and observe the fruit fly's nervous system in detail.Hampel et al. have now described the neural circuit that triggers and controls the stereotyped leg movements that the fruit fly Hampel et al. first identified sensory neurons in the flies' antennae that were needed to elicit grooming in response to irritating displacements of the antennae. Once these neurons were found, techniques—including those that allow specific neurons in the fly's brain to be precisely controlled—were then used to find other neurons that participate in the grooming process. This approach highlighted three groups of interneurons: two in the brain and one in the fly's equivalent of the spinal cord. Together these layers of sensory neurons and interneurons formed a circuit that triggered grooming whenever the antennae were disturbed.Notably, activating different sensory or interneurons triggered bouts of antennal grooming of differing durations. This shows that the same neural circuit can both produce highly specific movements and modify the movements to provide flexibility. Neural circuits with similar features have been observed previously to induce other animal behaviors, for example, swimming in leeches. This suggests that this organization may be common in circuits that elicit movements. Additional experiments are now needed to validate whether similar circuits underlie other stereotyped movements in fruit flies and other animals.DOI:http://dx.doi.org/10.7554/eLife.08758.002 An animal may perform a particular movement in response to its environment and internal state, and many movements are selected from a repertoire of stereotyped motor patterns. This repertoire can include movements that serve particular purposes, such as feeding, grooming, song production, locomotion, and even coordinated facial poses for expressing different emotions . Many ofThe proximal trigger of pattern generator activity arises from the command-like neurons, which can consist of individual neurons, or populations of neurons that induce or ‘command’ specific movements . Such neDrosophila has led to rapid progress in identifying command-like neurons. This progress has been enabled by the use of optogenetic and thermogenetic activation of specific, genetically targeted populations of neurons in freely moving adult flies  can be studied to determine the neuronal mechanisms by which specific movements are initiated and controlled. Grooming is ubiquitous among limbed animals as a means of protecting the body surface from different types of mechanical or chemical irritants . Such seWe previously discovered that activating different neuronal populations in the fly nervous system could induce distinct grooming movements, such as grooming of the eyes, antennae, wings, thorax, or legs . This raGiven that grooming is induced by stimulation of the body surface, we first sought to identify sensory neurons that could relay such stimulation from the antennae to the brain. To this end, we revisited the GAL4 lines we identified in our previous behavioral screen and examined them for expression in the antennae . One linWhile our results were consistent with these sensory neurons being responsible for eliciting antennal grooming, the expression of GAL4 in other neurons allowed the possibility that they were contributing to this behavior. To examine whether the JO neurons elicit antennal grooming, we used the intersectional Split GAL4 (spGAL4) approach in an effort to restrict GAL4 activity to these neurons . SpecifiConfocal imaging confirmed that each spGAL4 pair targeted neurons within the second antennal segment, as revealed by staining for the neuronal protein Elav  Kamikou. The anaThe JO comprises different subgroups of chordotonal neurons whose axons project to distinct zones (zones A–E) in the brain antennal mechanosensory and motor center (AMMC) . aJO axoGiven that aJO neurons project to zone C/E before passing to the SEZ, we tested whether activation of previously described C/E neurons could elicit antennal grooming. Indeed, activation of zone C/E neurons using published GAL4 drivers  elicitedThe muscles that control front leg movements necessary for antennal grooming are innervated by neurons residing in the most anterior region of the ventral nervous system (VNS), the prothoracic neuromeres (ProNm)  Burrows. BecauseOf the GAL4 lines that elicited antennal grooming in our previous screen , two lacTo validate that the spGAL4 lines target interneurons, we removed sensory neurons by amputating the antennae, and then measured the time spent grooming during thermogenetic activation. As anticipated, amputation did not abolish movements directed towards the antennal region, and some pairs significantly increased such movements relative to intact antennae . aBN1-spGAL4-1 targeted expression to aBN1, a single interneuron in the ventral brain . Two othThe other interneuron class comprises aDNs that project from the brain to the VNS. Five spGAL4 pairs targeted single descending neurons with cell bodies located in the posteroventral SEZ . Given tThe identified neuronal classes all have projections in the ventral brain . aBNs prWe first asked whether the grooming elicited by aJO thermogenetic activation required the activity of the aBNs and aDNs by performing a behavioral epistasis test in which we genetically silenced the activity of the interneurons while activating the aJO . For silWe next sought to identify a sensory stimulus upstream of this putative grooming circuit. Because JO neurons detect antennal movements , we reasWe next examined the functional necessity of the different interneuron classes for grooming in response to antennal displacement. aBN1 was found to be necessary, as expression of TNT in aBN1-spGAL4-1 or aBN1-spGAL4-2 both reduced the grooming response  that project within this ventral brain region, and descending neurons (aDNs) that project both within the ventral brain and descend to the VNS . These nHow does activation of this circuit elicit grooming of the antennae? Grooming movements are ultimately produced by pattern generators in the VNS that control the front and hind legs . The aDNIn this work, we examined a role for the identified circuit in detecting displacements of the antennae and eliciting grooming. This was based both on our discovery that the JO induces grooming, and that different JO neurons can detect antennal movements to elicit specific behavioral responses . FurtherAnother function of grooming is to remove debris, parasites, or other substances from the body surface . We haveA scenario whereby different types of stimuli, such as antennal deflections or dust can cause grooming could explain some of the complexity of the circuit that we observe. Our data suggest multiple possible routes of feedforward excitation through the circuit layers to induce antennal grooming. For example, the function of at least a subset of the aBN2 neurons was not necessary for grooming in response to physical displacement of the antennae, whereas they were necessary in response to thermogenetic activation of the JO. Thus, feedforward excitation likely has multiple routes through the antennal grooming circuit, but why would this be? One possibility is that each route reflects specific features of the sensory stimulus. Different stimuli might be sensed by distinct JO neurons or mechanosensory bristle neurons on the antennae that engage different ensembles of neurons within the circuit to produce specific grooming responses. Therefore, it may be revealing to test whether different stimuli coopt specific circuit components to fine–tune parameters of grooming, such as short vs persistent grooming durations.The finding that the antennal grooming circuit can induce persistent grooming raises the question as to its biological role. Our results do not reveal a natural stimulus that induces persistence, as displacements of the antennae rarely induced persistent grooming . PersistaJO mechanosensory neurons are critical for the grooming response to antennal movements and sufficient to induce grooming. This reveals a new role for the JO, which was previously implicated in hearing, gravitaxis, and wind-induced suppression of locomotion . These bOur data indicate that JO-induced antennal grooming is likely restricted to neurons projecting to zone C/E. Previously characterized JO neurons projecting to this zone are less sensitive to smaller movements of the antennae and adapt slowly to mechanical stimuli . The antTwo different populations of C/E-projecting neurons can induce antennal grooming; one that projects only to the AMMC, and the other (aJO) that projects to the AMMC and SEZ. This raises the question of whether both populations interact with the antennal grooming circuitry described in this work. The aBNs and aDN1 have neurites within the AMMC where they could plausibly receive excitatory inputs from both C/E neuron populations. Additionally, because neurons within zone C/E are implicated in other behaviors like wind-induced suppression of locomotion, it remains to be determined whether these JO neurons are multifunctional, or whether specific subpopulations within this group are responsible for distinct behaviors. Future work will be required to determine how these two populations of JO sensory neurons interact with the antennal grooming circuitry.The antennal grooming circuitry consists of at least three different neuronal classes. As our extensive screening efforts identified multiple GAL4 lines for each class, we presume that we have uncovered a major portion of the neurons that elicit antennal grooming. Indeed, these neurons are sufficient to form a functionally connected circuit that extends from the JO to the VNS. However, additional descending neurons are likely involved given that TNT expression in the aDNs failed to disrupt the grooming response to aJO activation. We also found evidence for an unidentified neuron downstream of aBN2 that inhibits aDN2.command-like neurons that evoke specific movements   . aBN1 anry input . Howeverd system . HoweverTritonia diomedia) and in the leech , trigger neurons immediately downstream of sensory neurons induce persistent swimming motor patterns   . Gating The layered and complex organization of the antennal grooming circuit raises the question as to its function. We discussed above the possibility that the circuit may have different possible routes of feedforward excitation. Such organization may provide different points at which the circuit can be modulated, thus allowing for flexible control of different movement parameters. In the antennal grooming circuit, flexible control of movement duration could be provided by the parallel aDNs , which aThe aDNs are also impinged upon by feedforward excitation and inhibition , possiblThe persistent grooming induced by brief excitation of the aBNs provides another mechanism for controlling grooming duration . This isIn this section we have discussed how the complex organization of the antennal grooming circuit might control movement duration, however, it could also control parameters that our current analysis methods cannot detect. The level of behavioral analysis presented in this work reveals that the induced trajectories of the legs are specific for the antennae rather than other head parts; however, it does not allow for the detection of finer antennal grooming movements. Grooming is characterized by an initial targeting of the legs to the stimulated body part, followed by cyclic movements that groom the region . Higher Recent work indicates that command-like neurons may constitute a common means of eliciting specific movements in insects. Tools that allow for both acute control of neuronal activity and precise genetic targeting of specific neurons in fruit flies have enabled experiments that merge behavioral analyses with real time neuronal manipulations. Such experiments demonstrate that activation of specific neurons can elicit distinct movements, such as escape, locomotion, or courtship song . AdditioFlies were reared on cornmeal and molasses food at 21°C and 50% relative humidity on a 16/8 hr light/dark cycle. 5–8 day old males were used for all experiments, except for those in UAS-dTrpA1 and screened for increased antennal grooming at 30–32°C. For pattern refinement and co-expression studies, the enhancers of GAL4 lines that exhibited increased antennal grooming were used to generate spGAL4 and LexA reagents, which were constructed as described previously  . To ideneviously  and prodeviously  used to 2 intensity , which flies cannot see. For CsChrimson activation, we used 656-nm red light at 27 mW/cm2 intensity (Mightex). The red light stimulus parameters were delivered using a NIDAQ board controlled through Labview . Red light frequency was 5 Hz for 5 s (0.1-s on/off), and 30-s interstimulus intervals . Grooming movements were manually scored as previously described  and UV cured glue  and then left to recover for 12 hr. The presence of the powder on the antennae did not cause increased antennal grooming after the recovery period. Flies were tethered using a pin that was glued to their thoraces and then positioned on a 6 mm diameter air-supported ball  within aw1118; UAS-TNT; pBPGAL4U). The percent time that control flies groomed when the magnetic field was applied was measured at different frequencies and voltages to determine the optimal stimulus conditions  test to compare more than three genotypes with each other. Next we performed a post-hoc Mann–Whitney U test and applied Bonferroni correction.Dissection and staining was performed as previously described , with moFor Antibodies used: rabbit anti-GFP , chicken anti-GFP , mouse anti-GFP , mouse mAb anti-nc82 , rat anti-DN-cadherin , rabbit anti-RFP , rat anti-flag , rabbit anti-HA , mouse anti-V5 , AlexaFluor-488 , AlexaFluor-568 , AlexaFluor-633 .https://www.nitrc.org/projects/cmtk/)   and asses/cmtk/)  a suite http://fiji.sc/). Image preparation, analysis of overlap, and adjustment of brightness and contrast were performed with Fiji  with an excitation wavelength of 920 nm. Imaging fields of view were chosen as to contain a distinctive process of the candidate post-synaptic neuron, which included processes at the midline for the aDNs and aBN2, and vertical running processes close to the midline for aBN1.Male flies were dissected in saline containing 103 mM NaCl, 3 mM KCl, 5 mM TES, 8 mM trehalose dihydrate, 10 mM glucose, 26 mM NaHCO2 and 800 μW/mm2. Trains were delivered at 50 Hz. Experiments usually started with a 50-pulse train. If no response was observed, the power was raised progressively until one occurred or the maximum power was reached. Each experimental run consisted of 4 repeats lasting for approximately 20 s. Runs were repeated approximately every 2 min. When postsynaptic responses were observed, we did not see any desensitization. See CsChrimson was excited with 2 ms pulses of 590-nm light via an LED shining through the objective. Instantaneous powers measured out of the objective ranged between 50 μW/mmhttp://julialang.org/).ΔF/F0 was calculated  for each video in a region of interest (ROI) obtained as follows: the average projection of the video was calculated and pixels of the projection were clustered by a k-means algorithm between ROI and non-ROI pixels. Of note, the selection method relies only on average intensity and not activity, because we wanted to use the same detection method for responsive and non-responsive runs. This also relies on selecting fields of view that unambiguously contain only the neuron of interest. Analysis was run in Julia ( review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing Editor, and three reviewers.Thank you for submitting your work entitled “A neural command circuit for grooming movement control” for peer review at The reviewers have discussed the reviews with one another, and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Drosophila. For example, one of the reviewers said: “The authors have done an impressive job in leveraging the state-of-the-art genetic/neural circuit tools in Drosophila to determine which neurons are necessary/sufficient for this behavior and how they functionally connect with each other. Moreover, they have a nicely supported model for functional interactions in the circuit, including the role of inhibition – this is terrific!”. Another reviewer commented: “This study will be highly interesting for neuroscientists in the field of motor control as well as for scientists studying animal behavior in general”. A third reviewer also stated: “The authors identify novel chordotonal sensory neurons innervating Johnston's Organ (JO) in the second antennal segment, and 3 classes of interneurons involved in this behavior, including 2 classes of local neurons (LNs) and several classes of descending neurons (DNs). This clearly required a heroic effort, and the authors are to be congratulated for their work”.All reviewers agree that this is an extremely thorough characterization of the neural circuits underlying an interesting behavior in Remarkably, optogenetic activation of any of these classes of neurons drives grooming of the antenna, and not of other body parts (at least during photostimulation) . This obIn summary, the authors have taken an important step forward in deconstructing a circuit that may ultimately explain a fascinating problem in sensory-motor coordination. They are encouraged to downplay the reciprocal excitation/persistent activity part of their story, which is the weakest, and emphasize the aspects of their findings that are most relevant to the grooming system, namely the remarkable functional specificity they have uncovered. The circuit architecture they have defined certainly hints at a more complex function and regulation than would be expected from a simple feed-forward stimulus-response reflex pathway, and this is interesting as well. Above all, they should make clear to the reader  the limitations of the methods they have employed, and the parts of the story that are rock-solid vs. those that are less so.Essential revisions:1) Which sensory neurons of the JO induce grooming?1A) It is unclear what distinguishes antennal movements for song, wind, and gravity perception  and antennal movements for grooming. Is it a much larger displacement of the antenna? Or is it the context? The authors show a plot where they increase the strength of the magnetic field (measured in volts) and show more grooming. They could easily measure the displacement of the antenna (using high speed video or a laser doppler vibrometer) and determine what range of antennal displacements (measured in nm) leads to grooming – this information is critical for knowing what strength of the stimulus activates the antennal grooming circuits. This is important because the receptor neurons for their behavior are likely overlapping with the receptor neurons for song, wind, and gravity detection, as the authors mention.1B) The authors interpret their findings to that effect that a specific subpopulation of JO neurons is exclusively responsible for providing sensory information specifically for antennal grooming. I agree that activation of these sensory neurons (SN) shows that they are sufficient to induce grooming. This, however, might be a general and unspecific stimulation of the JO which, in turn, elicits a general grooming response. You show in 1C) The authors show that the activity of several  of the classes of neurons they have described is required for this behavior. That is reassuring, at one level. However inspection of the video suggests that the magnitude of the antennal displacements (while not explicitly measured) produced by the magnet must be far greater than those normally produced by the application of dust. This raises the important question of whether the neurons described here are required for dust-evoked grooming. The authors do not comment on this point, and should. Presumably, they have tried this experiment, and apparently it did not work (otherwise why would they have gone to the trouble of constructing the elaborate magnet-evoked assay). The authors need to be up front with their readers about this, raise the issue and discuss it. There may be an interesting reason for the discrepancy. For example, is the artificial stimulus used here actually mimicking the antennal displacements produced by grooming behavior itself, rather than by the small particles that typically evoke grooming? If so, could the circuit uncovered here be part of a mechanism for feedback-control of grooming behavior? There is some hint of this, briefly touched on by the authors in passing, because amputation of the antenna actually causes an increased level of grooming behavior produced by thermogenetic activation of some of the interneuron classes. In any case, in the interests of full disclosure the authors need to discuss this important issue.2) The Introduction is not broad enough. For example, the authors need to provide more background on CPGs, patterned motor behaviors, and grooming, and to place the behavior they are studying in the context of the larger body of work. For example, the authors only cite papers on stick insect walking when introducing stereotyped motor patterns – they need to invoke behaviors like song production, feeding, etc. and compare/contrast their behavior with other well-studied motor patterns. In addition, the authors also don't provide enough background on their previous study  – this s3) Where do individual aJOs project? The authors don't report on any single cell clones to show that individual aJOs innervate all 3 brain regions mentioned  or that individual aJOs only innervate one region. If this experiment was not possible for some technical reason can the authors indicate this in the text of the manuscript?4) Functional connectivity versus synaptic connectivity: The data presented clearly show functional connectivity between neurons. However, there is only weak evidence (it is not ‘likely’), that functionally connected neurons are postsynaptic to each other since synaptic delays are known to be small relative to the temporal resolution of the GCaMP6s used. Their approach detects functional connectivity but not synaptic connectivity – which is fine. However, the authors should make this clear throughout the manuscript (and in the Abstract).Drosophila at higher resolution . In other words, there is much more to get out of this system now that the circuit is mapped.5) Resolution of the behavior: Given the authors' approach to scoring the behavior manually  it is not possible to examine whether activity of different neurons in the antennal grooming circuit leads to different types/aspects of behavior or different timing of the behavior or differences in trial-to-trial reproducibility. Given that the authors suggest in their Introduction and Discussion that this behavior is a good model for understanding the contribution of sensory drive and modulation to stereotyped motor programs, it would help to know how stereotyped the behavior is. The authors should add a paragraph to their Discussion of the future value of examining the grooming behavior of 6) The differentiation of the classes of antennal grooming descending neurons (aDN) appears to be less specific compared to the two aLN-classes. For example, no information is given on aDN2 in 7) In the third paragraph of the subsection “Connectivity among the antennal grooming neuronal classes”, based on the neuroanatomical analysis of the branching patterns of the described sensory and brain interneurons, the authors conclude specific connectivity between them. I agree that their data show that these neurons are likely to belong to the same circuit, but I do not see proof of an explicit synaptic connectivity between them: (i) the anatomical analysis of the branching patterns has not the scale to allow for that – a scale of 25µm as given in the figures is not sufficient to resolve connectivity e.g. see  and (ii)8) With regards to the subsection “A command circuit that elicits variable grooming durations”, all neural circuits are composed of “multiple neurons”. It is not clear which point the authors want to make. Specifically, information is lacking on how the new findings relate to previous findings on neurons that serve command-like functions in insect behavior  and in the fruit fly? With respect to the fruit fly I was missing a discussion on what the new study adds to the previous work on command neurons in the control of fruit fly courtship behavior e.g. , aversiv9) Is there really a reciprocal excitation loop in this circuit, and is it responsible for persistent grooming behavior evoked by optogenetic stimulation? The authors place a great deal of emphasis on the finding of an apparent reciprocal excitation loop between the aLN1 and aLN2 local interneurons , and inv10) Furthermore, the magnitude of the average LN1 response to “LN2 stimulation” in 11) In addition, if the authors really want to make the case that this putative reciprocal excitatory loop is responsible for the persistent grooming behavior they observe, then they should perform an epistasis experiment in which they demonstrate that persistent grooming triggered by aLN2 activation (but perhaps not grooming per se) is blocked by inactivating aLN1 cells, and/or vice-versa.Finally, the authors should point out that in their antennal displacement assay, only a small fraction of stimulation trials elicited persistent grooming, and that even in those cases the persistence appears to be in the grooming of the eyes/head, not the antenna . So the  […] In summary, the authors have taken an important step forward in deconstructing a circuit that may ultimately explain a fascinating problem in sensory-motor coordination. They are encouraged to downplay the reciprocal excitation/persistent activity part of their story, which is the weakest, and emphasize the aspects of their findings that are most relevant to the grooming system, namely the remarkable functional specificity they have uncovered. The circuit architecture they have defined certainly hints at a more complex function and regulation than would be expected from a simple feed-forward stimulus-response reflex pathway, and this is interesting as well. Above all, they should make clear to the reader  the limitations of the methods they have employed, and the parts of the story that are rock-solid vs. those that are less so.We thank the reviewers for their kind and thoughtful comments regarding this work. The manuscript is revised to better emphasize the discovery of a circuit that induces a highly specific grooming movement. The possible role of reciprocal excitation as the cause of persistent grooming is now deemphasized, but the manuscript retains some focus on the circuit as a potential mediator of grooming duration. We have added additional experiments related to the antennae and Johnston’s organ requested by the reviewers, including two full figure supplements for Essential revisions:1) Which sensory neurons of the JO induce grooming?Responses to specific queries for question 1 are addressed independently , and 1C). At the end, these responses are assembled into a cohesive conclusion. Additionally, because question 3 also fits nicely with this question, we incorporated our response to it with question 1 conclusions.1A) It is unclear what distinguishes antennal movements for song, wind, and gravity perception  and antennal movements for grooming. Is it a much larger displacement of the antenna? Or is it the context? The authors show a plot where they increase the strength of the magnetic field (measured in volts) and show more grooming. They could easily measure the displacement of the antenna (using high speed video or a laser doppler vibrometer) and determine what range of antennal displacements (measured in nm) leads to grooming – this information is critical for knowing what strength of the stimulus activates the antennal grooming circuits. This is important because the receptor neurons for their behavior are likely overlapping with the receptor neurons for song, wind, and gravity detection, as the authors mention.We measured how far the antennae are displaced when the electromagnet was driven at two different voltages (5 and 10V). Five volts induces limited grooming whereas ten volts induces near the maximum amount of grooming . The meaThe measurement of antennal displacement magnitude is now in the Results text and incorporated into 1B) The authors interpret their findings to that effect that a specific subpopulation of JO neurons is exclusively responsible for providing sensory information specifically for antennal grooming. I agree that activation of these sensory neurons (SN) shows that they are sufficient to induce grooming. This, however might be a general and unspecific stimulation of the JO which, in turn, elicits a general grooming response. You show inthat silencing aJO neurons significantly reduces grooming; while this suggests that aJO is involved in eliciting grooming it only shows sufficiency, not necessity. Also, this is still consistent with a general decrease of JO sensitivity. Therefore, the following questions need to be addressed to support the conclusion that these SNs are specific for grooming: (i) did activation of the other previously described subpopulations of JO SNs  fail to induce grooming?We tested whether activation of previously described JO subpopulations induces grooming. Excitation of zone C/E neurons using previously published GAL4 drivers elicits antennal grooming, whereas excitation of zone A/B neurons did not (new data added as (ii) If aJO neurons are indeed the sole JO neurons necessary for grooming then ablation of all non-aJO neurons should not result in a decrease in grooming behavior.Our results indicate that expression of TNT in the aJO almost completely blocks the grooming response to imposed movements of the antennae . Therefo(iii) It may have slipped my attention, but was the role of antennal exteroceptors, e.g. tactile hairs, tested? All of these points have to be addressed before you can definitely say that aJO neurons are necessary and sufficient for grooming, i.e. part of the grooming circuit. As it stands now, you have shown that grooming can be induced by stimulating at least part of the JO.The bristle mechanosensory neurons on the antennae have not been tested for their role in grooming because we have not yet acquired spGAL4 lines that can target them. We added text to address the bristle mechanosensory neurons in the Discussion .1C) The authors show that the activity of several  of the classes of neurons they have described is required for this behavior. That is reassuring, at one level. However inspection of the video suggests that the magnitude of the antennal displacements (while not explicitly measured) produced by the magnet must be far greater than those normally produced by the application of dust. This raises the important question of whether the neurons described here are required for dust-evoked grooming. The authors do not comment on this point, and should. Presumably, they have tried this experiment, and apparently it did not work (otherwise why would they have gone to the trouble of constructing the elaborate magnet-evoked assay). The authors need to be up front with their readers about this, raise the issue and discuss it. There may be an interesting reason for the discrepancy. For example, is the artificial stimulus used here actually mimicking the antennal displacements produced by grooming behavior itself, rather than by the small particles that typically evoke grooming? If so, could the circuit uncovered here be part of a mechanism for feedback-control of grooming behavior? There is some hint of this, briefly touched on by the authors in passing, because amputation of the antenna actually causes an increased level of grooming behavior produced by thermogenetic activation of some of the interneuron classes. In any case, in the interests of full disclosure the authors need to discuss this important issue..” In brief, the large deflections that induce grooming described in this paper are likely to be caused by non-debris stimuli such as static electricity or unexpected displacements of the antennae. We cite several papers that support this idea. We also address the role of this circuitry in the removal of debris, such as dust from the antennae. Different sensory neuron types other than the JO neurons may play a role in detecting debris on the antennae and inducing grooming . However, we have not yet found lines that specifically target the bristle neurons to test this possibility.We discuss this in the first two paragraphs of a new subsection of the Discussion called “Biological functions of the antennal grooming circuit3) Where do individual aJOs project? The authors don't report on any single cell clones to show that individual aJOs innervate all 3 brain regions mentioned  or that individual aJOs only innervate one region. If this experiment was not possible for some technical reason can the authors indicate this in the text of the manuscript?We added a multicolor stochastic labeling experiment to the manuscript. This revealed that the majority of neurons within the aJO have similar projections. Most of the cells project from the AMMC to the ventral SEZ, and a smaller subset projects from the AMMC to the posterior SEZ (new data added as Conclusions for questions 1 and 3:A) Activation of JO neurons targeted by previously described GAL4 lines that express in the different JO regions revealed that C/E-projecting neurons induce grooming, whereas A/B-projecting subtypes do not (new data added as B) We find evidence that at least two subsets of zone C/E JO neurons induce grooming. The previously described C/E neurons show no obvious projections to the SEZ, but instead terminate in the AMMC . In contC) The antennal displacement distance that induces grooming (31 +/- 6 μm) is within the range shown to induce calcium responses in previously described C/E JO neurons. Therefore, aJO neurons might also be activated by such large displacements, coupled with our finding that they are critical for the grooming response to these displacements.Overview of major revisions:A) We revised and extended the Results and Discussion to incorporate the new results and draw the conclusions mentioned above. Particularly, we added text to the Results section (last two paragraphs) called “A group of Johnston’s Organ mechanosensory neurons elicits antennal grooming” and Discussion section, “A distinct class of JO neurons detects antennal displacements and elicits grooming”.B) Because there are likely two populations of zone C/E JO neurons that induce grooming, we no longer say aJO neurons are responsible for antennal grooming in the Abstract. Instead we state, “Mechanosensory chordotonal neurons detect displacements of the antennae and excite three different classes of functionally connected interneurons, which include two classes of brain interneurons and different parallel descending neurons.”C) Given that additional zone C/E neurons induce grooming, and that aJO-LexA may target some of these non-aJO neurons , we now 2) The Introduction is not broad enough. For example, the authors need to provide more background on CPGs, patterned motor behaviors, and grooming, and to place the behavior they are studying in the context of the larger body of work. For example, the authors only cite papers on stick insect walking when introducing stereotyped motor patterns – they need to invoke behaviors like song production, feeding, etc. and compare/contrast their behavior with other well-studied motor patterns. In addition, the authors also don't provide enough background on their previous study  – this study is in many ways a follow-up, but they expect the reader to have familiarity with this paper already.Drosophila, and additional information on our previous work. However, on the latter point, our previous work was primarily focused on how grooming is organized into a sequence of movements, which is not what we intended this paper to be about. The only point that we want to introduce from this previous work is the finding that activating different subsets of neurons induces specific grooming movements. This sets up the identification of neurons that specifically elicit antennal grooming. We have added two additional sentences to the last paragraph of the Introduction that more completely describes this point from our previous work. Additionally, the reviewers asked us to compare and contrast grooming with other stereotyped motor patterns. We felt that this was better addressed in more detail in the Discussion, framed in the context of what we learned from the results presented in the paper. The last section of the Discussion discusses the antennal grooming circuit in the context of other command-like neurons that have been described in flies. We hope that the reviewers are satisfied with this approach.We now provide additional background information on stereotyped movement patterns, CPGs, the study of neurons that induce specific movements in 4) Functional connectivity versus synaptic connectivity: The data presented clearly show functional connectivity between neurons. However, there is only weak evidence (it is not ‘likely’), that functionally connected neurons are postsynaptic to each other since synaptic delays are known to be small relative to the temporal resolution of the GCaMP6s used. Their approach detects functional connectivity but not synaptic connectivity – which is fine. However, the authors should make this clear throughout the manuscript (and in the Abstract).We made numerous changes throughout the manuscript to make clear that our evidence supports functional connectivity among the neurons. Where appropriate, we now use the terminology suggested by the reviewers, such as “neurons downstream” or “neurons upstream” (suggested in question 7). In other places we now refer to the neurons as “functionally connected” rather than connected.5) Resolution of the behavior: Given the authors' approach to scoring the behavior manually  it is not possible to examine whether activity of different neurons in the antennal grooming circuit leads to different types/aspects of behavior or different timing of the behavior or differences in trial-to-trial reproducibility. Given that the authors suggest in their Introduction and Discussion that this behavior is a good model for understanding the contribution of sensory drive and modulation to stereotyped motor programs, it would help to know how stereotyped the behavior is. The authors should add a paragraph to their Discussion of the future value of examining the grooming behavior of Drosophila at higher resolution . In other words, there is much more to get out of this system now that the circuit is mapped.We have added a paragraph to the Discussion that now addresses this important point. In brief, we discuss that the antennal grooming circuit might control parameters that our current analysis methods cannot detect. The level of behavioral analysis presented in this work does not allow for the detection of fine antennal grooming movements. Thus, higher resolution analysis of the kinematics of the leg movements may allow for testing the roles of the circuit in controlling different variables of antennal grooming. This text can be found in the last paragraph of the Discussion called “A command circuit that elicits variable grooming durations.”6) The differentiation of the classes of antennal grooming descending neurons (aDN) appears to be less specific compared to the two aLN-classes. For example, no information is given on aDN2 inand its legend. In general throughout the text the number of DN-classes that are referred to is changing from two , to one  and to three . In order to justify the summary, it is important that the authors allow the reader to keep clear track for all data available on DN classes leading to this scheme.We made several revisions to the text to ensure that our logic for stating that there are three aDNs is consistent and clear throughout. First, we added text to the last sentence of the last paragraph of the Results section called “Three different interneuron classes elicit antennal grooming” that should help establish that there are three aDNs. This text reads: “However, we did not identify a spGAL4 combination that exclusively expresses in the third R18C11-targeted aDN (named aDN3)” Second, we added text to the 7) In the third paragraph of the subsection “Connectivity among the antennal grooming neuronal classes”, based on the neuroanatomical analysis of the branching patterns of the described sensory and brain interneurons, the authors conclude specific connectivity between them. I agree that their data show that these neurons are likely to belong to the same circuit, but I do not see proof of an explicit synaptic connectivity between them: (i) the anatomical analysis of the branching patterns has not the scale to allow for that – a scale of 25µm as given in the figures is not sufficient to resolve connectivity (e.g. see) and (ii) the same holds for the calcium transients recorded (e.g. see). Such explicit interpretation is not justified. A terminology like “neurons downstream” or “neurons upstream” is appropriate.See question 4 for the revisions that we made to the manuscript. Of note, we incorporated the reviewers’ suggestion to use terminology, such as neurons upstream or downstream.8) With regards to the subsection “A command circuit that elicits variable grooming durations”, all neural circuits are composed of “multiple neurons”. It is not clear which point the authors want to make.We agree with the reviewers that we did not sufficiently describe the significance of circuits that consist of multiple neurons. We clarified this point in the third paragraph of the subsection “A command circuit that elicits variable grooming durations” (in the Discussion). We describe how the antennal grooming circuit and other well-described circuits are organized such that some neurons induce movements that persist beyond their initial activation, whereas other neurons must be continually activated to continue the movement. We point out that this type of organization appears to be common among sensory-connected neuronal circuits that induce specific movements.Specifically, information is lacking on how the new findings relate to previous findings on neurons that serve command-like functions in insect behavior  and in the fruit fly? With respect to the fruit fly I was missing a discussion on what the new study adds to the previous work on command neurons in the control of fruit fly courtship behavior (e.g.), aversive motor behavior  and walking direction ?We added a new section to the Discussion entitled “Neurons that command specific movements in insects.” This section describes how work in flies and other insects suggests that the use of dedicated neurons to command specific movements is a common mechanism of behavioral control in insects. We then insert the caveat that these command-like neurons may be embedded in larger, more dynamic neural networks in which neurons that elicit one movement also participate in other movements. We cite work from leech that supports this possibility.9) Is there really a reciprocal excitation loop in this circuit, and is it responsible for persistent grooming behavior evoked by optogenetic stimulation? The authors place a great deal of emphasis on the finding of an apparent reciprocal excitation loop between the aLN1 and aLN2 local interneurons , and invoke it to explain their interesting observation that activation of these interneurons can produce persistent grooming . Unfortunately, due to limitations in the specificity of the reagents used to perform these experiments, the evidence of reciprocal excitation is unconvincing and it is very difficult to correlate the physiological (imaging) and behavioral data.Questions 9, 10, and 11 all relate to the strength of evidence supporting aBN1/aBN2 reciprocal functional connections. We address the specific points for each of these questions and then summarize the revisions related to all of them at the end.Firstly, the evidence of reciprocal connectivity is weak. The degree of anatomical overlap between aLN1 and aLN2 neurons is very limited . Although the evidence for functional connectivity from LN1→LN2 neurons is convincing , the evidence for the reciprocal connection  is not. Apparently (based on the information provided in), to test for the latter connection the authors used an LN2-LexA driver to express CsChrimson in these neurons (in contrast to the reverse experiment where they used an LN1-GAL4 driver to express the opsin). This LN2-LexA driver is extremely broadly expressed, in the central brain and optic lobes . Without specific 2-photon targeted activation of the LN2 neurons themselves, it is difficult to be confident that the excitatory drive on the LN1 neurons inis indeed due to input from the aLN2 cells.The reviewers requested that we used dashed outlines to mark the different projection regions. We realized with this comment that there was confusion about the meaning of AMMC, posterior and ventral SEZ in this context. We did not intend for these to refer to distinct regions of the neuropile, but to the three major regions defined by the aJO projections shown in We agree that the aBN2-LexA driver expresses in additional neurons, raising the possibility that aBN2 is not the upstream cause of the aBN1 increased calcium response shown in 10) Furthermore, the magnitude of the average LN1 response to “LN2 stimulation” inis much weaker than that of the LN1 → LN2 stimulation in. Close inspection of the supplemental data indicates that even this weak average recording is misleading, because only a fraction of the flies tested, and only a small fraction of trials, yielded any response at all . This is very different from a result in which the neurons showed a weak response in most of the flies in most of the trials. This is all the more puzzling given that there are more aLN2 than aLN1 cells , and that the activation of neurons in the aLN2-spGAL4-2 line has the strongest effect to promote grooming of any of the lines characterized in the paper . Thus, the evidence for an LN2→LN1 connection that is relevant to grooming is, in my opinion, weak and unconvincing.We revised the manuscript to more completely describe the data that contributes to the average traces shown in At the very least, the circuit diagram inshould be re-drawn to show that the connection from aLN2→aLN1 is far weaker and more questionable than the reverse connection (perhaps using a dashed line and question mark), and this issue and associated caveats should be clearly spelled out in the Discussion.11) In addition, if the authors really want to make the case that this putative reciprocal excitatory loop is responsible for the persistent grooming behavior they observe, then they should perform an epistasis experiment in which they demonstrate that persistent grooming triggered by aLN2 activation (but perhaps not grooming per se) is blocked by inactivating aLN1 cells, and/or vice-versa.The manuscript is now revised to deemphasize the putative reciprocal excitation between aBN1 and aBN2. We describe the caveats associated with the experiment to test the aBN2 to aBN1 functional connectivity. The major revisions are described below.Description of major revisions for questions 9-11:A) We removed mention of the reciprocal excitation from the Abstract.B) We added description of the caveats associated with the aBN2 to aBN1 functional connectivity experiment, and explicitly state that the connection is only weakly supported by the data. This description is found in the fourth paragraph of the Results section called “Functional connectivity among the antennal grooming neuronal classes.” As the reviewers pointed out, we now indicate that only high-intensity red light activation of aBN2 could cause a weak calcium response in aBN1. We mention that this response was inconsistent, as only three out of five flies tested showed increases in calcium in aBN1. We further point out that the aBN2-LexA driver also expresses in neurons in other parts of the brain (discussed above).C) We removed most of the discussion of reciprocal excitation as an underlying mechanism causing the persistence. However, reciprocal excitation is briefly mentioned in the sixth paragraph of the Discussion section called “A command circuit that elicits variable grooming durations” as one of two possible mechanisms that could cause the persistence. This text reads: “Although our work only provides weak evidence for reciprocal excitation between the aBNs, a mechanism whereby reciprocal connections maintain their excitation offers a plausible explanation for the persistence that we observe. Alternatively, aBN1 and aBN2 could have intrinsic membrane properties that allow them to produce prolonged responses to brief excitation. Thus, future experiments will examine whether reciprocal excitation or intrinsic membrane properties of the aBNs produce persistent grooming .”Finally, the authors should point out that in their antennal displacement assay, only a small fraction of stimulation trials elicited persistent grooming, and that even in those cases the persistence appears to be in the grooming of the eyes/head, not the antenna . So the biological relevance of this persistence phenomenon, interesting though it may be, is not obvious.The fourth paragraph of the Discussion section entitled “Biological functions of the antennal grooming circuit” addresses the possible biological relevance of persistent grooming. We discuss how our results do not identify a natural stimulus that induces reliable persistent grooming. However, persistence was previously described in the grooming responses of other animals, where it was shown to be at least partially dependent on the nature of the stimulus . Thus, future work will explore the possibility that specific stimulus parameters can evoke persistent antennal grooming in flies.


The purpose was to study hearing aid (HA) use in persons 65 years and older, and to investigate how socioeconomic and hearing related factors were associated to use of HA.This study included 11,602 persons (65 years and above) from the second Nord-Trøndelag Health Study (HUNT2) and the integrated North-Trøndelag hearing loss study (NTHLS) in 1995–1997. Audiometry was taken of all participants. Missing information about use of HA in possible users of HA existed in data from 1103 (9.5 %) of the participants. Effects of sociodemographic variables, low, medium and high frequency hearing thresholds and being bothered by their hearing were explored in men and women, adjusting the effects for each other. Cross tabulations and logistic regression analyses were used.In all, 14 %  were users of HA, but 62 % had a mean hearing impairment (HI) based on 0.5, 1, 2, 4 kHz over both ears >25 dB. Use of HA was associated with higher education. Adjusting for all covariates and hearing variables, each 10 dB medium frequency threshold shift increased the chance of HA-use by a factor of two-three in both men and women. Having reported being bothered by hearing loss additionally increased the chance around sevenfold. Low frequency hearing thresholds were not associated with HA-use in women. In men, low frequency hearing thresholds up to 50 dB increased odds for use of HA, but low frequency hearing thresholds ≥ 70 dB decreased odds for use of HA. Men living with a spouse had higher odds for using HA compared to men without a spouse. For women there is no difference between those with and without spouse in use of HA. Men and women without spouse did not differ in their use of HA.About two third of 65 years and older participants had a HI higher than 25 dB, but only one seventh used HA. Use of HA was associated with higher than basic education. Men without a spouse were less likely to use HA compared to men with a spouse. Hearing impairment (HI) in older adults (65 years and above) is one of the most common chronic health conditions in the western world today, and the prevalence of HI increases with age . HI has HI creates a significant burden for the persons who suffer from it . It may Older adults may have need of audiological rehabilitation, including fitting of HA, but do not seek treatment or do not accept use of HA to improve their hearing. There are barriers for both fitting HA –22 and fUse of HA has in most clinical audiological studies been found to be positively associated with older persons’ experience of their hearing loss, i.e. those who experience more severe hearing difficulties and are the most bothered by their loss are more likely to use HA after fitting . FurtherThe study aimed to assess the prevalence of hearing aid use in older adults (65 years and more), and to investigate how socioeconomic and hearing related factors such as degree of HI and being bothered by HI were associated with use of hearing aid.The second wave of the Nord-Trøndelag Health Study took place in 1995–1997 (HUNT2). All inhabitants aged 20 years or older residing in Nord-Trøndelag County were invited to participate. In HUNT 2 93,898 persons were invited, 65,237 participated (69.4 %). HUNT 2 included as an integrated project the Nord–Trøndelag Hearing Loss Study (NTHLS) . The priIn many aspects Nord-Trøndelag is considered fairly representative of Norway , but it has no large cities and a somewhat lower level of education than the national average.The study has been approved by the Norwegian Regional Committee for Medical Research Ethics.Audiometry in HUNT2. Air- conduction hearing thresholds were obtained by pure tone audiometry with Interacoustics AD25 automatic self-administered audiometers with TDH-39 earphones linked to a personal computer (PC). Data were automatically stored at the PC after testing. A few persons were offered a manual audiometry because they were not able to follow the instruction for the automatic procedure. Bone conduction thresholds were not examined and masking was not used. Otoscopy was carried out prior to the audiometry by two professional audiologists in a sub-sample with 6415 subjects. 10.9 % of the ears were observed with abnormal pinna or external ear or with abnormal tympanic membrane and 2.5 % were observed with ear canal obstruction, and the rest were considered normal [d normal . These cThe standard frequencies included in the tests were 250, 500, 1000, 2000, 3000, 4000, 6000, and 8000 Hz. Thresholds were determined in accordance with ISO 8253-1 (1989) . Mean heThe audiometers were calibrated every six month. Semi-portable, dismountable sound attenuation booths were used in rooms specially selected to avoid background noise. Background noise was measured on a random sample and met the recommended standard for test administration  . FurtherUse of HA was observed by questionnaire. Those who reported that they knew they had a hearing loss, were asked to answer the following question: Do you use hearing aid? (Yes/no.) If responding “no” to having a hearing loss, missing data on hearing aid use were treated as no hearing aid. If confirming to have a hearing loss or not responding to this question, and if the measured mean hearing threshold value was lower than 26 dB, missing data on the hearing aid item were treated as no hearing aid (n = 599). Otherwise participants with missing hearing aid data were excluded (n = 1103).Being bothered by hearing loss was assessed with one self-report item in HUNT 2, questionnaire 1 (Q1). Those who reported that they knew they had an impaired hearing, were asked how much bothered they were by this impairment, either a little (1), moderately (2), or a lot (3). Subjects who did not report a hearing impairment in Q1 were scored 0.Demographic information includes information about marital status and highest level of education. Marital status gives information of whether the participant has a spouse or not. The highest educational level achieved reported in HUNT 2 (Q1) was scored according to the OECD guidelines for classification [fication . The oriData were analyzed by use of SPSS version 22.0 .The main outcome, using HA (versus not), was studied using logistic regression analysis for men and women separately. Preliminary analyses were run entering the independent variables low, medium and high frequency thresholds as categorical variables with every 10 dB as a separate category. The results showed an almost linear effect for medium and high frequency thresholds, that is, a similar increase in odds ratios (OR) for every 10 dB change in hearing threshold along the whole distribution for medium and high frequency thresholds. Thus, medium and high frequency thresholds were entered as continuous variables scaled with 10 dB as units for men and women. A linear increase in OR from 20 dB for every 10 dB change in low frequency hearing threshold was found up till 70 dB, but not thereafter. Other variables of interest for use of hearing aids explored in unadjusted analysis were age, marital status, education and being bothered by hearing loss. Age was in the preliminary analyses found to be linearly associated with the outcome and was entered as a continuous variable in the analyses. In all regression analyses the lowest score was set as reference group whenever possible.Interaction effects between hearing thresholds and age, marital status and education on hearing aid use were first checked separately in men and women, and so were interaction effects between age, marital status and education. Lastly, interaction effects were studied in the total sample, also entering the interaction terms HI X gender.P-values ≤ 0.05 were considered statistically significant.In the first models sociodemographic variables and measured hearing thresholds in low, medium and high frequencies were included. In the second model an additional variable, being bothered by hearing loss were included. The results were reported as OR with 95 % confidence intervals (CI). Off all participants, 62 % 6480 of 10,499) had a mean HI  higher than 25 dB and 14 %  used HA. From the latter 889 (60.4 %) were men . No other interaction test reached significance.In the fully adjusted logistic regression model , we found use of hearing aid to be associated with higher education, higher medium frequency hearing thresholds, and being bothered by hearing loss in both men and women. The results are shown in Table To the best of our knowledge, this is the first study which has assessed association between sociodemographic and hearing related variables - including measured HI and self-reported hearing loss - and use of HA in a large population-based sample of older persons. In all, less than one seventh (14 %) of the participants used HA, but about two third had a HI  higher than 25 dB over both ears. Use of HA was associated with higher degree of HI, being bothered by hearing loss and education above ground level in both men and women. Men without a spouse were less likely to use HA compared to men with a spouse. Women without spouse were not more or less likely to use HA than women with spouse or men without a spouse.The prevalence of HA-use among community-dwelling older adults was in the present study equally low as other population-based studies 8–13 %) , 10, 12.3 % , 10,It is expected that the need to improve hearing with use of HA increases as the degree of HI worsens. Even so, two review reports indicate that the evidence of an association between higher degree of HI and increasing chance of using HA after an audiological HA fitting process is mixed , 24. In Furthermore, as expected and previously found in clinical audiological studies and population-based studies, experienced consequences of HI were positively associated with HA-use , 22, 27,This large scale population study made it possible to study interaction between independent variables for HA-use, and we found an interaction between gender and marital status. Men having a spouse were twice as likely to be HA-users compared to men without a spouse, but there were no difference in use of HA between women with and without a spouse. We did not study the participants’ communication demands or the role of significant others in the present study, i.e. the spouse of the participants or other close relatives. However, others have found the communication demands and the role of significant others to influence audiological help seeking and/or HA-use , 36. WomThe strengths of this study are mainly the large study sample  and the objective measure of HI. The participants were aware of this study's specific hypotheses during data collection, which limits the risk of reporting bias of HA-use.The limitations of the present study need to be addressed. Even if the effects of the various audiological variables were adjusted by sociodemographic variables, we cannot rule out that there are confounders which we have not adjusted adequately for. In addition to communication demands and role of significant others, which we did not have information about, personality and cosmetic demands may influence HA acquiring and later HA-use , 24, 37.http://www.n-t-a-f.org/HA-statistikk.htm). That does not necessarily show that the real HA use has been doubled, since we have a higher HI prevalence in the nation due to the ageing of the population, people may change HAs more frequently today  than twenty years ago and that it is more usual to choose HA for both ears now than 20 years ago, but it may reflect a higher proportion of HA users. But even if better technology may have caused a somewhat increased occurrence of HA-use during the last two decades, most of the reasons for using or not using HA have hardly changed fundamentally. Unfortunately, absence of more recent audiometrical data connected to use of HA makes it difficult to know the extent to which our results are valid for today’s hearing aid use.There is usually some uncertainty related to the generalizability of results from population studies like ours. The hearing examination represented only a small part of the total examination program in the HUNT 2 study, so there is little reason to suspect that hearing acuity has substantially influenced the recruitment to the study. A reduction of the study sample due to missing data on single questionnaire items may have affected the estimated occurrence of HA-use somewhat, but has hardly more than trivially affected the observed relationships between the predictors and the outcome. Nord-Trøndelag county is considered to be quite representative of Norway, so there is also little reason to doubt that these results are valid nationwide. The extent to which the results can be generalized internationally is more questionable. It seems reasonable to assume that the results are essentially valid at least for rather wealthy societies with refund arrangements for HA similar to the Norwegian. A more important limitation to the generalizability is the age of the data, especially because the HA technology has developed considerably since 1998. Statistics on number of acquired HA per year have more than doubled in Norway since the time of our data collection, from 39 778 in 1998 to 84 606 in 2015 (The proportion of HA-users, 14 % among people aged 65 years or older, was low considering the much larger proportion with HI. Use of HA was strongly associated with higher degree of HI along most of the distribution of HI. Being bothered by hearing loss was a very strong indicator of HA-use even after adjusting for measured HI. Education above ground level also was asociated with HA-use in both men and women. Men without a spouse were less likely to use HA compared to men with a spouse, whereas HA-use in women did not depend on marital status.CI, confidence intervals; dB, decibel; HA, hearing aid; HI, hearing impairment; kHz, kilo Hertz; OR, odds ratio