Obesity poses a significant risk of developing type II diabetes and other diseases. Hedgehog (Hh) signaling has been shown to inhibit adipose tissue development, but its effect on diet-induced obesity during postnatal life is not known. Here by inducing expression of constitutively active Smoothened (SmoM2) or Gli2 (ΔNGli2) in the adipocyte lineage of postnatal mice, we show that targeted activation of Hh signaling suppresses high-fat-diet-induced obesity and improves whole-body glucose tolerance and insulin sensitivity. Both SmoM2 and ΔNGli2 induce the expression of Wnt6, a known anti-adipogenic factor, in fat depots of the mouse. Hh-Gli2 signaling inhibits not only adipocyte differentiation but also lipogenesis in adipocytes in vitro. Finally, pharmacological inhibition of Porcupine, an acyltransferase essential for Wnt secretion, alleviates both anti-adipogenic and anti-lipogenic effects of Hh in cell culture models. Overall, targeted activation of Hh signaling ameliorates diet-induced obesity and may be explored for pharmaceutical development. The global epidemic of obesity affects hundreds of millions of people worldwide in 2016, as estimated by the World Health Organization. In the United States, one third of the adult population is obese . It is wHedgehog (Hh) signaling is an evolutionarily conserved pathway controlling tissue development and homeostasis. In this pathway, Hh ligands bind to the receptor Patched 1 (Ptch1) to relieve its inhibition on Smoothened (Smo), a seven-pass transmembrane protein, resulting in transcriptional activation by the Gli family of transcription factors . HoweverHh signaling has been implicated in the development of adipose tissues. Genetic studies in Drosophila identified Hh as a potent inhibitor of fat body formation . DeletioFabp4  promoter resisted fat accumulation  from the drinking water. To this end, we first assessed the tissue specificity and efficacy of Pparg-tTA by generating mice with the genotype of Pparg-tTA;TetO-Cre;mT/mG and monitoring GFP expression in response to Dox withdrawal. After two months of Dox withdrawal starting at two months of age, we observed GFP in both the gonadal fat  and the interscapular fat depot  as well as the bone marrow fat, but not in the liver, the intestine or the heart . HoweverSmoM2/+) on regular chow plus Dox from conception through two months of age, at which point they either continued on Dox (+Dox) or were weaned off Dox (-Dox) for up to 8, 18 or 26 weeks  raised on regular chow and Dox from conception through two months of age were subjected to a high-fat diet (HFD) for up to 16 weeks, with or without continued Dox treatment   . The –Doctively) . At 8 wectively) . Both thctively) . In addictively) . Moleculctively) . Moreovectively) . Finallyctively) . To testctively) . After 8ctively) . Thus, HAlthough Hh signaling has been shown previously to inhibit adipogenesis, the underlying mechanism is not fully understood. To gain additional insights, we studied the anti-adipogenenic effect of Hh signaling in the murine bone marrow mesenchymal progenitor cell line M2-10B4 (hereafter M2), which we have previously shown to respond robustly to Hh signaling . The M2 ΔNGli2/+ mice, and activated expression of ΔNGli2 (a constitutively active form of Gli2) from the Rosa26 locus with an adenovirus expressing Cre (Ad-Cre)  . ΔNGli2 (Ad-Cre) . Thus, GΔNGli2/+ were maintained on Dox and regular chow from conception to two months of age before being separated into two groups, with both on HFD but one continuing on Dox (+Dox) and the other off dox (-Dox) for up to 18 weeks. The –Dox mice was significantly leaner than their +Dox counterparts after 8 weeks of HFD, and their difference in body weight increased with time  for 72 hr . Those eh Ad-GFP . Moreoveh Ad-GFP . The indh Ad-GFP . Importah Ad-GFP . The faiΔNGli2/+ mice that had been maintained on Dox since conception (no ΔNGli2 expression), and then cultured the cells for 3 days in either growth media or adipogenic media with (+Dox) or without Dox (-Dox). As expected, the –Dox cells expressed more Gli1 mRNA but responded significantly less to the adipogenic stimuli that induce Pparg and Fabp4  is essential for Wnt secretion, we used IWP2, a small molecule inhibitor of Porcn, to inhibit paracrine Wnt signaling . In M2 cnd Fabp4 . Howevernd Fabp4 . Thus, iΔNGli2/+ mice. Specifically, we induced adipocyte differentiation for three days and then activated ΔNGli2 expression with Ad-Cre in the insulin-only media with or without IWP2 for eleven days. Whereas expression of ΔNGli2 induced by Ad-Cre reduced the average size of lipid droplets, IWP2 abolished the effect  and then assessed the effect of PM specifically on the lipid-accumulating phase (eleven days in insulin-only media). RT-qPCR assays indicated that PM significantly reduced the expression of lipogenesis genes such as Lpl (lipoprotein lipase), Fasn (fatty acid synthase), Plin (perilipin) and Dgat1 (diacylglycerol acyltransferases 1) without affecting the differentiation marker Fabp4 . Furthere effect . Thus, b14C6]-glucose to the insulin-only media for the final three days of the lipid-accumulating phase  with or without the addition of PM or IWP2. PM significantly decreased the 14C contribution to lipid whereas IWP2 had the opposite effect. Importantly, IWP2 restored glucose contribution to the control level in the presence of PM even though the rescued level was lower than that by IWP2 alone  were maintained in RPMI-1640 (Gibco) with 10% fetal bovine serum (Gibco) as per ATCC instructions. The cell line was authenticated with the CO1 assay (interspecies) and tested free of mycoplasma contamination with Hoechst DNA stain (indirect) and Agar culture (direct) by ATCC. Unless otherwise indicated, M2 cells were seeded at the density of 1 × 10protocol . Adipogeprotocol . For knoprotocol . The tarΔNGli2/+ mice, the cells were first induced for differentiation for 3 days and then incubated with the insulin-only medium containing either Ad-GFP or Ad-Cre and with or without the supplementation of IWP2 with media change every 3–4 days. The ImageJ software was used to measure the size of lipid droplets stained positive by oil red O. 100 droplets were measured for each treatment in each experiment and three independent experiments were conducted. Representative results from one experiment were presented here. IWP2 (Sigma) was used at 5 μM dissolved in DMSO. PM  was used at 1 μM dissolved in DMSO.Preadipocytes were isolated as the stromal vascular fraction (SVF) from the gonadal fat pad according to a published protocol . AdipogeTotal RNA was isolated with QIAGEN RNeasy kit  and transcribed into cDNA with iScript cDNA synthesis kit (Bio-Rad). Fast-start SYBR Green (Bio-rad) was used for qPCR in Step-One machine (ABI). Nucleotide sequence of primers is listed in 14C6] glucose  was added at 0.2 μl/ml to the medium, with or without PM or IWP2, for three more days. Lipids were extracted with Lipid Extraction Kit , and an equal volume of the extract was measured either with Lipid Quantification Kit  or for radioactivity with a scintillation counter. The amount of 14C radioactivity was normalized to lipid quantity (cpm/μg). Glucose and lactate measurements were done as described . The minimal sample size of 3 was calculated according to http://www.stat.ubc.ca/~rollin/stats/ssize/n2.html, with 25% difference in mean values, 10% standard deviation and the default α (0.05) and power (0.80) values.All quantitative data are presented as mean ±SD with a minimum of three independent samples. Statistical signiﬁcance is determined by Student’s t test or two-way factorial ANOVA  in tissues other than adipose tissue. This inhibitory effect appears to be mediated by the Hh downstream effector, Gli2, likely through activation of the Gli2 target gene, Wnt6. These are significant findings of general interest.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work as it stands will not be considered further for publication in eLife policy to only invite revisions in cases where revisions can be reasonably completed within two months, we cannot invite revision of the current manuscript. However, we are in principle interested in the study and we would be enthusiastic about reviewing a substantially revised version that addresses our concerns, with the proviso that it will be treated as a new submission. Such a submission would not necessarily be seen by the same reviewers and thus additional concerns might be raised. eLife is highly selective, which means that the majority of submissions are rejected, but we thank you for sending your work for review and we hope you will submit to eLife again in the future.However, despite the interesting set of experiments, the paper has several weaknesses relating to the Dox treatments and alleles used, level of modification of Wnt singaling, and presentation which we anticipate will require extensive reanalysis and the generation of additional data. Since it is Reviewer #1:Gli2, the principal transcriptional activator used by Hh signaling  and inhibition of Wnt secretion modestly reverses the Gli2-mediated inhibition of adipogenic gene expression in vitro . In addition to affecting adipogenesis, Hh pathway activation in vitro has mild effects on fat formation in those adipocytes that do form .In this manuscript, Shi et al. use a conditional approach to activate Hh signaling in a subset of adult WAT and BAT cells or their precursors . Hh pathignaling . Gli2 isGli2 as the main effector of pathway activation in the fat.Several previous studies have demonstrated that the Hh pathway is a strong anti-adipogenic pathway including two in vivo studies, in which adipose-specific Hh activation completely inhibits WAT  and BAT  formation. This study complements these previous data nicely by suggesting that Hh activation can also inhibit diet-induced obesity in adult mice. This group then carefully analyzed the downstream cascade and convincingly argue for Major concerns:The Ppary-tTA allele used here has been shown to cause lipodystrophy in the absence of Dox . Moreover, Dox treatment suppresses these pathologies, suggesting that the observed lipodystrophy results from promiscuous transcriptional activity of tTA. Since the phenotype reported in this manuscript is that fat mass is reduced in the absence of Dox, an alternative explanation could be that as soon as Dox is removed, the anti-adipogenic effects of this allele are activated, thereby counteracting the fat mass gain due to the high fat diet. To separate their findings from the anti-adipogenic role of the Ppary-tTA line, the authors should repeat the experiment with mice just carrying the Ppary-tTA allele {plus minus} Dox.Similarly, many different classes of antibacterial agents, including tetracyclines, promote animal growth. Subtherapeutic levels of chlortetracycline causes increased adiposity and metabolic alterations in mice . Since the phenotype reported in this manuscript is that fat mass is reduced in the absence of Dox, an alternative explanation could be that removal of Dox removes this recognized pro-adipogenic influence. To separate their findings from the pro-adipogenic role of Dox, the authors should repeat the experiment with mice lacking one of the three transgenic alleles {plus minus} Dox.Given that Wnt6 is induced by Hh signaling 17-fold in M2 cells, 1500-fold in MEFs but only 3-fold in vivo , and IWP2 has a modest effect (less than twofold on Pparg expression) on preadipocytes, and certainly doesn't restore the robust inhibition of adipogenesis caused by purmorphamine , it isn't clear that Wnt signaling is the primary means by which Hh signaling inhibits adipogenesis. Some confirmation of the effect on Wnt signaling in vivo by Western of animal tissue and analysis of Wnt signaling in fat pads would lend important support to this crucial point. Similarly, is Wnt6 induced in the SmoM2 model, as one would expect?Reviewer #2:Gli2. These authors further showed that both differentiation and lipid accumulation were inhibited by Hh signaling, likely through activation of a Gli2 target gene, Wnt6. Wnt6 expression is dependent on Hh signaling in M2 cells, MEFs, and adipose tissue. Blocking palmitoylation of Wnt6 reduced Wnt signaling activity and alleviated the inhibitory effect of Hh signaling on adipogenesis.That activation of Hh signaling inhibits adipogenesis (both in white and brown adipose tissue) is well established in cultured cells and in mice. However, whether this can protect mice from obesity and its related metabolic complication has not yet been reported. In this paper, Shi and Long presented a comprehensive set of in vivo and in vitro data to show that adipose tissue specific activation of Hh signaling in postnatal mice not only reduces adiposity but also improves glucose tolerance and insulin sensitivity in diet-induced obese mice. This inhibitory effect is mainly carried out by the Hh downstream effector, As far as I know, this is the first paper to provide genetic evidence that activation of Hh signaling in mice can have a direct effect on diet-induced obesity. Because Hh signaling can inhibit adipogenesis in cultured cells, it's no surprise to see that mutant mice with high Hh pathway activity in adipose tissue are leaner even under high fat-diet treatment. However, the fact that these mice also showed significant improvement of glucose tolerance and insulin sensitivity is impressive and suggests that adipose tissue specific activation of Hh can affect glucose utilization  in tissues other than adipose tissue. In my view, this is the most important finding from this study and needs to be further characterized in this paper. For example, gene expression analysis of skeletal muscle, liver, or subcutaneous adipose tissue would provide insight into the expression of genes responsible for glucose uptake and insulin pathway activation in these tissues. Therefore, it is somewhat disappointing that the authors focused on characterizing the effect of Hh on inhibiting adipogenesis, which has been previously reported by several studies rather than on how Hh regulates glucose tolerance and insulin insensitivity through controlling adipose tissue formation in high-fat diet fed mice.Gli2. However, the in vitro differentiation assays appear not to be working very well. In all the cell lines  examined, adipogenic induction (control cells) only resulted in the differentiation of a low% of cells, making it difficult to draw definitive conclusions about the extent to which differentiation is disrupted because most of the cells did not respond to the adipogenic induction. Perhaps adjusting the concentration of the adipogenic induction media components will in the first place improve the efficiency of differentiation and provide more definitive results to support the author's conclusions.Overall, the data presented in this paper is well organized and easy to understand, and the genetic data is convincing and nicely shows the epistatic relationship between Smo and Reviewer #3:Gli2, at the transcriptional level. In ChIP-seq experiments in a cell culture system, they find that Gli2 binds to several Wnt loci, and knockdown of Gli2 prevents Hedgehog stimulated activation of these Wnts. The pharmacological inhibition of Wnt proteins largely (but not completely) prevents Hedgehog-mediated reduction of adipogenesis in cultured cells, suggesting that Wnts are the major mediator of Gli2-mediated suppression of adipogenesis. This model is consistent with their earlier data suggesting that Hh-repression occurs cell autonomously.In this study, Shi and Long report that activation of Hedgehog pathway reduced the development of obesity among mice fed a high fat diet. This repression occurs through This study is interesting and logically presented but I do have one major concern. The connection between Hedgehog signaling and Wnt activation, a major conclusion of the study, seems somewhat contrived, relying on 'cherry-picked' ChIP binding data that (see below) and cell culture experiments with different Gli and Wnt kinetics that are hard to compare with the in vivo models. Clarifying the transcriptional relationship between Hedgehog and Wnts in these systems is critical for solidifying this connection.1) As I understand it, the current data suggest that Hedgehog inhibits adipogenesis but whether it would cause an already obese mouse to lose weight remains unaddressed. While I realize that doing this experiment is beyond the scope of the study, I raise this concern because the authors are making the claim that Hedgehog pathway activation could potentially be used to treat obesity. If my understanding is correct, the authors should consider toning-down / or qualifying statements like "Thus, targeted activation of the Hh pathway may be explored to combat diet-induced obesity."2) Why is the magnitude of Hh-mediated response so different between high fat and regular diets for SmoM2? Both Gli1 and Ptch1 have higher responses in the RosaSmoM2 and RosaGli2deltaN dominant active genetic systems compared to the SmoM2 in mice maintained on a normal diet. Is this a confounding biological factor?3) Related to point #2, it is not clear in Figure 9D how long pre-adipocytes were cultured without DOX before assaying by qRT-PCR. There, the difference in Gli1 induction (4-fold) is really striking compared to the in vivo Gli1 response using RosaGli2deltaN system, which appears at least 25-fold higher c.f. . This maGli2-deltaN activated mice have no upregulation Wnt 5a or Wnt 9a. While they do have significant upregulation of Wnt 6  it is much lower than the values obtained in the cell culture assays. Would this level of Wnt really be sufficient to account for cell autonomous Gli-mediated repression? Is this discrepancy due to negative feedback from prolonged Wnt signaling, that the in vitro systems are not effectively modeling the in vivo response, or that Wnts are, despite the ChIP data, not directly regulated by Gli2 in the in vivo systems?4) The fat pads from the activated Gli2 ChIP data provided for review is incomplete and does not meet current standards for providing all raw data. The ChIP binding coordinates are not described in the excel file that is provided, the raw data (ChIP-seq reads) must be deposited in GEO with an accession number and reviewer access provided for reviewer / editorial access. In addition, the currently provided dataset  should list the ChIP coordinates. The methods for analyzing the ChIP data need to be clarified. For example, what does the P-value in the table refer to? Are these peaks enriched relative to an input or to a cell line not expressing Flag? Are enhancers for known Hedgehog pathway targets  also bound by Gli2?5) The 6) Besides the known roles for Wnts in inhibiting adipogenesis, it is not clear why the focus is on the three Wnt genes for the ChIP experiment. In my quick examination of the predicted target genes provided in the excel spreadsheet using an online KEGG enrichment analysis tool, the most upregulated pathways were , Notch (13 genes), Tgf-β (16 genes), Wnt (19 genes). This cursory analysis suggests that a strong rationale for focusing exclusively on Wnt needs to be provided. A confounding factor in the current level of data analysis is that, if my understanding is correct, the nearest gene in the genome is shown on the dataset regardless of whether it is transcriptionally active in adipocytes. Could the ChIP regions be intersected with Hedgehog-responsive genes in adipocytes (or if this is unavailable at least genes known to be transcribed in adipocytes) to predict which of the ChIP regions are functional?7) Related to concern 4, I am concerned that these Wnt sites may not represent direct Gli targets. The ability the Gli-bound sites to act as enhancers should be tested in cell culture enhancer assays in the presence or absence of Hedgehog pathway activation.8) Please indicate the genetic background of the mice in the experiments.9) Are there sex-specific differences in Hh-mediated inhibition of obesity? If so, they should be shown. If not, this should be stated.10) Figure 9A-C: What is the time course of the Wnt inhibition experiments? Please provide appropriate details in the Materials and methods / Results sections.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]Gli2 prevents obesity induced by high fat diet in adult mice" for further consideration at eLife. Your revised article has been evaluated by Fiona Watt (Senior editor) and two reviewers, one of whom evaluated the previous version.Thank you for resubmitting your work entitled "Hedgehog signaling via Summary:Gli2 shows Wnt6 induction. Primary preadipocytes isolated from gonadal fat pad of PPAR ΔNGli2/+ and in the presence of doxycycline and IWP2 induce Pparg and Fabp4 adipocyte markers. This is an interesting study that the authors have strengthened since the original submission.The study by Long and Shi offers novel insights into a role of Hh signalling in adult adipose tissue physiology and diet-induced obesity. Although a mouse model used in the study provides limited efficiency in targeting different fat depots, it shows strong anti-adipogenic and anti-lipogenic effects of Hh signalling activation in high-fat diet condition. The lineage tracing experiments using Pparg-tTA;TetO-Cre in combinations with R26-SmoM2 and R26-ΔNGli2/+, separately, are both convincing of a SHH mediated effect on adult adipogenesis, high-fat diet induced obesity and suppressing metabolic dysfunctions as assessed by glucose tolerance and insulin sensitivity. The in vitro data using primary adipocytes is also convincing of SHH-mediating Wnt induction. RNA from whole gonadal fat pad from both transgenic lines overexpressing SHH or Essential revisions:Gli2 regulates Wnt activity, they do not provide compelling support the direct transcriptional regulation of Wnt proteins by Gli2.The major concern remains the link between Hedgehog signalling and Wnt activation, since the results do not strongly support the authors' conclusions. These concerns are expanded in the specific comments below. While the data strongly suggest that Gli2. I can think of other approaches that could be used to test this (in vivo transgenic enhancer assays or possibly CRISPR-based deletions that are clearly outside the scope of the current work and might ultimately end up showing no enhancer activity.1) The enhancer experiments shown in Gli2 at the Wnt6 region compared to binding at Gli1 or Wnt9a . This, combined with the fact that the ChIP data was acquired from a lentivirally driven Gli2 construct in M2 cells does not provide high levels of confidence that these represent Gli2 binding in adipocytes.2) The ChIP-seq data in Gli2 functions at least partly through direct transcriptional regulation of several Wnt proteins"; Discussion section "Further downstream, several Wnt genes are direct transcriptional targets of Gli2." Both of these statements convey the erroneous perception that Gli22 directly regulates multiple Wnt proteins. Their own data suggests that Wnt5a and 9b may not be in vivo targets . Thus, at best, it could be claimed that Gli2 regulates the transcriptional regulation of a Wnt protein. However, the evidence that Gli2 regulates Wnt6 is unconvincing.3) Discussion section "Furthermore, 4) The new supplemental spreadsheet showing ChIP-seq reads is helpful. However, both within the supplemental dataset and subsection “ChIP-seq experiments”, there is no statistical information on the statistical analysis done for peak calling . Similarly, in the supplemental spreadsheet, there is a 'scaled fold change'. While it is not mentioned how this was calculated, it seems like it is a modified read count in dox treated versus dox untreated. The authors should calculate a modified P-value to reflect the quality of these peaks.5) The authors need to tone down their claims of a direct transcriptional link between Shh and Wnt, and remove the data that the reviewers consider to be weak (see above).Gli2 prevents obesity induced by high fat diet in adult mice" for further consideration at eLife. Your revised article has been evaluated by Fiona Watt (Senior editor) and the two original reviewers.Thank you for resubmitting your work entitled "Hedgehog signaling via The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below. Specifically, the reviewers request that you remove all the ChIP-seq data from the main body of the paper and the abstract. In addition, since none of the in vitro assays appears to be adipogenic, the manuscript lacks a solid connection between the in vivo findings and the downstream molecular mechanism studies and you should address this within the text.As highlighted previously there are several problems with your ChIP-seq data: 1) there are many binding regions that do not have enhancer activity – so this binding does not necessarily imply that the regulation is direct, and 2) the ChIP-seq results were obtained in M2 cells. [Editors’ note: the author responses to the first round of peer review follow.][…] Reviewer #1:[…] The Ppary-tTA allele used here has been shown to cause lipodystrophy in the absence of Dox . Moreover, Dox treatment suppresses these pathologies, suggesting that the observed lipodystrophy results from promiscuous transcriptional activity of tTA. Since the phenotype reported in this manuscript is that fat mass is reduced in the absence of Dox, an alternative explanation could be that as soon as Dox is removed, the anti-adipogenic effects of this allele are activated, thereby counteracting the fat mass gain due to the high fat diet. To separate their findings from the anti-adipogenic role of the Ppary-tTA line, the authors should repeat the experiment with mice just carrying the Ppary-tTA allele {plus minus} Dox.We appreciate the reviewer’s concern and have now repeated the experiment with mice just carrying the Pparg-tTA allele as recommended. Specifically, the Pparg-tTA mice were raised with Dox from conception to 2 months of age, and then fed HFD with or without Dox water for 2 additional months. We have found no Dox effect on body composition, body weight or GTT. Please see the data in the supplemental figures .Similarly, many different classes of antibacterial agents, including tetracyclines, promote animal growth. Subtherapeutic levels of chlortetracycline causes increased adiposity and metabolic alterations in mice . Since the phenotype reported in this manuscript is that fat mass is reduced in the absence of Dox, an alternative explanation could be that removal of Dox removes this recognized pro-adipogenic influence. To separate their findings from the pro-adipogenic role of Dox, the authors should repeat the experiment with mice lacking one of the three transgenic alleles {plus minus} Dox.We have now repeated the experiment as suggested. Specifically, wild type mice without any of the transgenic alleles were raised with Dox from conception to 2 months of age, and then fed HFD with or without Dox water for 2 additional months. We detected no Doc effect on body weight or body composition .Given that Wnt6 is induced by Hh signaling 17-fold in M2 cells, 1500-fold in MEFs but only 3-fold in vivo , and IWP2 has a modest effect (less than twofold on Pparg expression) on preadipocytes, and certainly doesn't restore the robust inhibition of adipogenesis caused by purmorphamine , it isn't clear that Wnt signaling is the primary means by which Hh signaling inhibits adipogenesis. Some confirmation of the effect on Wnt signaling in vivo by Western of animal tissue and analysis of Wnt signaling in fat pads would lend important support to this crucial point. Similarly, is Wnt6 induced in the SmoM2 model, as one would expect?We agree that Hh signaling likely engages multiple downstream effectors besides Wnt6 to inhibit adipogenesis in vivo (see Discussion section). As per the reviewer’s suggestion, we have now performed additional experiments to confirm the involvement of Wnt signaling. Specifically, we show that a common Wnt target gene Axin2 was up-regulated in the gonadal fat pad upon ΔNGli2 expression . We furtReviewer #2:As far as I know, this is the first paper to provide genetic evidence that activation of Hh signaling in mice can have a direct effect on diet-induced obesity. Because Hh signaling can inhibit adipogenesis in cultured cells, it's no surprise to see that mutant mice with high Hh pathway activity in adipose tissue are leaner even under high fat-diet treatment. However, the fact that these mice also showed significant improvement of glucose tolerance and insulin sensitivity is impressive and suggests that adipose tissue specific activation of Hh can affect glucose utilization  in tissues other than adipose tissue. In my view, this is the most important finding from this study and needs to be further characterized in this paper. For example, gene expression analysis of skeletal muscle, liver, or subcutaneous adipose tissue would provide insight into the expression of genes responsible for glucose uptake and insulin pathway activation in these tissues. Therefore, it is somewhat disappointing that the authors focused on characterizing the effect of Hh on inhibiting adipogenesis, which has been previously reported by several studies rather than on how Hh regulates glucose tolerance and insulin insensitivity through controlling adipose tissue formation in high-fat diet fed mice.Gli2-Wnt6 signaling axis as an important part of the anti-adipogenic mechanism.We appreciate that the reviewer recognizes the significance of our finding. As the anti-obesity effect of Hh activation in the face of high fat diet is of potential translational value, we have focused our effort on gaining more mechanistic insights about the effect. The studies have uncovered an Hh-Besides the reduced adiposity, Hh activation also achieved impressive metabolic benefits in insulin signaling and glucose handling. We believe that the metabolic benefits are secondary to the reduced obesity but not necessarily specific to Hh signaling. We agree with the reviewer that a mechanistic understanding of the link between adiposity and whole-body metabolism is critical, but believe that such a fundamental question warrants a separate study.Overall, the data presented in this paper is well organized and easy to understand, and the genetic data is convincing and nicely shows the epistatic relationship between Smo and Gli2. However, the in vitro differentiation assays appear not to be working very well. In all the cell lines  examined, adipogenic induction (control cells) only resulted in the differentiation of a low% of cells, making it difficult to draw definitive conclusions about the extent to which differentiation is disrupted because most of the cells did not respond to the adipogenic induction. Perhaps adjusting the concentration of the adipogenic induction media components will in the first place improve the efficiency of differentiation and provide more definitive results to support the author's conclusions.We appreciate the reviewer’s concern. We think the relatively insufficient adipogenesis (judged by oil red O staining) of M2 cells may reflect their nature as bipotent mesenchymal progenitors for osteoblast and adipocytes. In addition, the adipogenic media that we used for M2 and MEFs did not contain troglitazone that could have produced more robust adipogensis. The primary preadipocytes however did exhibit robust differentiation in our assays, although we did not include images of oil red O staining in the paper for the sake of brevity. Regardless, in all cases in addition to quantifying the number of oil red O-stained cells, we have performed qPCR for several molecular markers to assess adipogenesis quantitatively . TherefoReviewer #3:[…] 1) As I understand it, the current data suggest that Hedgehog inhibits adipogenesis but whether it would cause an already obese mouse to lose weight remains unaddressed. While I realize that doing this experiment is beyond the scope of the study, I raise this concern because the authors are making the claim that Hedgehog pathway activation could potentially be used to treat obesity. If my understanding is correct, the authors should consider toning-down / or qualifying statements like "Thus, targeted activation of the Hh pathway may be explored to combat diet-induced obesity."We appreciate the reviewer’s concern and agree that future experiments are necessary to determine whether Hh activation is sufficient to reverse diet-induced obesity. We have deleted the statement in question.2)Why is the magnitude of Hh-mediated response so different between high fat and regular diets for SmoM2? Both Gli1 and Ptch1 have higher responses in the RosaSmoM2 and RosaGli2deltaN dominant active genetic systems compared to the SmoM2 in mice maintained on a normal diet. Is this a confounding biological factor?We thank the reviewer for the astute observation. We do not know for certain why this is the case at the moment. As targeting of the adipocytes by our strategy was < 50% in mice on the regular diet , it is p3) Related to point #2, it is not clear in Figure 9D how long pre-adipocytes were cultured without DOX before assaying by qRT-PCR. There, the difference in Gli1 induction (4-fold) is really striking compared to the in vivo Gli1 response using RosaGli2deltaN system, which appears at least 25-fold higher c.f. . This maGli2 in preadipocytes is less robust than that in the more mature adipocytes that are only present in the intact adipose depot. We acknowledge the limitations of the in vitro system, and that the in vivo anti-adipogenic effect of Hh signaling may be mediated by additional mechanisms besides Wnt induction.The preadipocytes were cultured with or without Dox for three days before assaying by qRTPCR. The relatively low induction of Gli1 compared to that in vivo could be due to the short duration of Dox withdrawal, or the small percentage of cells expressing Pparg-tTA due to cell heterogeneity in the preadipocyte preparation . It is also possible that the transcriptional response to 4) The fat pads from the activated Gli2-deltaN activated mice have no upregulation Wnt 5a or Wnt 9a. While they do have significant upregulation of Wnt 6  it is much lower than the values obtained in the cell culture assays. Would this level of Wnt really be sufficient to account for cell autonomous Gli-mediated repression? Is this discrepancy due to negative feedback from prolonged Wnt signaling, that the in vitro systems are not effectively modeling the in vivo response, or that Wnts are, despite the ChIP data, not directly regulated by Gli2 in the in vivo systems?Gli2 only in the preadipocytes that may be a small constituency of the adipose depot in vivo. As per the reviewer’s suggestion, we have now performed in vitro luciferase reporter assays to assess the Gli-bound sites in mediating the transcriptional activation by Hh signaling. The results show that two out of the four sites exhibit measurable activities  The Gli2 ChIP data provided for review is incomplete and does not meet current standards for providing all raw data. The ChIP binding coordinates are not described in the excel file that is provided, the raw data (ChIP-seq reads) must be deposited in GEO with an accession number and reviewer access provided for reviewer / editorial access. In addition, the currently provided dataset  should list the ChIP coordinates. The methods for analyzing the ChIP data need to be clarified. For example, what does the P-value in the table refer to? Are these peaks enriched relative to an input or to a cell line not expressing Flag? Are enhancers for known Hedgehog pathway targets  also bound by Gli2?Gli2 binding to both Ptch1 and Gli1 locus, with the latter now shown in We had up-loaded the raw data to the SRA database (BioProject: PRJNA374459) and provided the detailed methods for analyzing the ChIP-seq data in the revised Materials and methods section. We now also include a supplemental Excel file showing analyses results from both ChIP-seq and RNA-seq. The peaks enriched were relative to cells not induced with Dox and therefore not expressing Flag. We indeed see 6) Besides the known roles for Wnts in inhibiting adipogenesis, it is not clear why the focus is on the three Wnt genes for the ChIP experiment. In my quick examination of the predicted target genes provided in the excel spreadsheet using an online KEGG enrichment analysis tool, the most upregulated pathways were , Notch (13 genes), Tgf-β (16 genes), Wnt (19 genes). This cursory analysis suggests that a strong rationale for focusing exclusively on Wnt needs to be provided. A confounding factor in the current level of data analysis is that, if my understanding is correct, the nearest gene in the genome is shown on the dataset regardless of whether it is transcriptionally active in adipocytes. Could the ChIP regions be intersected with Hedgehog-responsive genes in adipocytes (or if this is unavailable at least genes known to be transcribed in adipocytes) to predict which of the ChIP regions are functional?Gli2 signaling”).We thank the reviewer for the constructive comment. We have previously generated a RNA-seq dataset in M2 cells after 72 hrs of purmorphamine treatment , and now provide a full list of genes with a minimum of 2-fold change in expression . By intersecting the RNA-seq and ChIP-seq data, we identified a list of candidate Hh target genes, these including three Wnt genes. Analyses of the candidate target genes with DAVID also identified Wnt signaling as a relevant pathway. We have revised the text to explain our thought process (subsection “Wnt6 is a direct target of Hh-7) Related to concern 4, I am concerned that these Wnt sites may not represent direct Gli targets. The ability the Gli-bound sites to act as enhancers should be tested in cell culture enhancer assays in the presence or absence of Hedgehog pathway activation.We appreciate the reviewer’s concern. We acknowledge that Wnt5a and Wnt9a may not be bona fide targets in vivo as they were not induced in either SmoM2 or ΔNGli2 mice . We have8) Please indicate the genetic background of the mice in the experiments.The mice were in a mixed genetic background of C57BL/6 (~70%) and 129 (~30%).9) Are there sex-specific differences in Hh-mediated inhibition of obesity? If so, they should be shown. If not, this should be stated.There is no sex difference in Hh-mediated inhibition of obesity. Data from either male or female are presented as stated in figure legends.10). Figure 9A-C: What is the time course of the Wnt inhibition experiments? Please provide appropriate details in the Materials and methods / Results sections.These data are now presented in [Editors' note: the author responses to the re-review follow.]Essential revisions:The major concern remains the link between Hedgehog signalling and Wnt activation, since the results do not strongly support the authors' conclusions. These concerns are expanded in the specific comments below. While the data strongly suggest that Gli2 regulates Wnt activity, they do not provide compelling support the direct transcriptional regulation of Wnt proteins by Gli2.1) The enhancer experiments shown in 2) The ChIP-seq data in 3) Discussion section "Furthermore, Gli2 functions at least partly through direct transcriptional regulation of several Wnt proteins"; Discussion section "Further downstream, several Wnt genes are direct transcriptional targets of Gli2." Both of these statements convey the erroneous perception that Gli22 directly regulates multiple Wnt proteins. Their own data suggests that Wnt5a and 9b may not be in vivo targets . Thus, at best, it could be claimed that Gli2 regulates the transcriptional regulation of a Wnt protein. However, the evidence that Gli2 regulates Wnt6 is unconvincing.4) The new supplemental spreadsheet showing ChIP-seq reads is helpful. However, both within the supplemental dataset and subsection “ChIP-seq experiments”, there is no statistical information on the statistical analysis done for peak calling . Similarly, in the supplemental spreadsheet, there is a 'scaled fold change'. While it is not mentioned how this was calculated, it seems like it is a modified read count in dox treated versus dox untreated. The authors should calculate a modified P-value to reflect the quality of these peaks.5) The authors need to tone down their claims of a direct transcriptional link between Shh and Wnt, and remove the data that the reviewers consider to be weak (see above).Gli2, but instead focus on Wnt signaling being induced downstream of Hh activation. Please note the major changes shown in red in the text. We deleted the enhancer experiment  there are many binding regions that do not have enhancer activity – so this binding does not necessarily imply that the regulation is direct, and 2) the ChIP-seq results were obtained in M2 cells.According to your recommendation, we have removed all the ChIP-seq data and the related text from the main body as well as the abstract. We believe the in vitro studies provide supportive evidence for the potential involvement of Wnt activation, and therefore have decided to keep them in the revision. At the same time, we acknowledge the limitation of such studies in both Results and Discussion sections, and state clearly that the in vivo relevance of such findings needs to be tested in vivo.


Transcranial direct current stimulation (tDCS) applied over the dorsolateral prefrontal cortex (DLPFC) has been shown to reduce cravings in tobacco addiction; however, results have been somewhat mixed. In this study, we hypothesized that motivation to quit smoking is a critical factor of tDCS effects in smokers. Therefore, we conducted a double-blind, randomized clinical trial to evaluate the effects of both tDCS and motivation to quit on cigarette consumption and the relationship between these two factors. DLPFC tDCS was applied once a day for 5 days. Our primary outcome was the amount of cigarettes smoked per day. We collected this information at baseline (d1), at the end of the treatment period (d5), 2 days later (d7) and at the 4-week follow-up (d35). Visual Analog Scale (VAS) for motivation to quit was collected at the same time-points. 36 subjects  were randomized to receive either active or sham tDCS. In our multivariate analysis, as to take into account the mediation and moderation effects of motivation to quit, we found a significant main effect of tDCS, showing that tDCS was associated with a significant reduction of cigarettes smoked per day. We also showed a significant interaction effect of motivation to quit and treatment, supporting our hypothesis that tDCS effects were moderated by motivation to quit, indicating that higher levels of motivation were associated with a larger tDCS response. We found that the participants' motivation to quit alone, both at baseline and at follow-up, does not explain the decrease in the average cigarette consumption. Repetitive prefrontal tDCS coupled with high motivation significantly reduced cigarette consumption up to 4-weeks post-intervention.http://ClinicalTrials.gov, NCT02146014.Clinical Trial Registration:  Smoking is the leading cause of preventable death worldwide and has been included in the International Classification of Diseases (ICD) of the World Health Organization (WHO) since 1992. It is considered to be a pandemic with one-third of the world population smoking, and is estimated to be the cause of more than 5 million deaths globally each year Bennett, . Like otnucleus accumbens (NAC) and to the prefrontal cortex (PFC). This system forms a cerebral gratification circuit with dopamine as the neurotransmitter, determining the sensation of pleasure associated with smoking  to the Indeed, techniques of neuromodulation targeting the prefrontal cortex can be used to reduce smoking and related behaviors. Previous transcranial Direct Current Stimulation (tDCS) studies have demonstrated the effectiveness of this technique in reducing the desire for smoking in active smokers  for at least 1 year, and being able to provide informed consent to participate. Exclusion criteria were as follows: being pregnant, having important clinical or psychiatric comorbidity that could interfere with the follow-up, being illiterate, or being treated for smoking at the time of enrollment.http://ClinicalTrials.gov under the number NCT02146014. All subjects participated voluntarily and signed the Informed Consent Form. The sample was recruited through flyers at the University of Brasília's Hospital (HUB), via dissemination on the university's website and local print media.The present study was approved by the Ethics Committee on Research in Human Beings of the Faculty of Health Sciences at the University of Brasília (CEP/FS/UnB), registry N: 080/10, deposited in the Registry System Thirty-six smokers participated in the study . Note that no sample size calculation was performed.This trial had two arms, active and sham tDCS, and the protocol consisted of a total of 7 visits. Each participant received active or sham tDCS once a day for five consecutive days day 1 to day 5). A computer-generated randomization sequence was used to assign the group, active or sham, in a 1:1 allocation ratio. Assessements were performed at baseline (d1), at the end of the stimulation sessions (d5), 2 days later (d7) and at 4 weeks follow-up (day 35); see Figure  to day 5A battery-powered direct current stimulator which uses two 9-Volt batteries for current generation and an additional 9-volt battery for an analog amperemeter display was used. The device has a sham mode switch and is capable of delivering from 0 to 4 mA through a pair of conductive rubber electrodes (anode and cathode) fitted with saline-soaked sponges.2 (7 × 5 cm), soaked in saline solution were used with the following montage: anode over the left DLPFC  and cathode over the right contralateral supraorbital region.Electrodes coated with sponges, measuring 35 cmDuring the experiment, subjects were sitting comfortably in an armchair in a bright and quiet room. The device was placed strategically behind the armchair, hidden from the patient during the stimulation. A constant current of 1 mA for 20 min was applied with a 10-s ramp-up at the beginning and a 10-s ramp-down at the end of the stimulation. For the sham tDCS, the same electrode montage was used with the same ramps-up and down of 10 s; however, the current was turned off after 20 s. A third person, not involved in the assessments or in data analysis, applied the stimulation. This procedure allowed both patients and assessors to be blinded to the treatement allocation.Our primary outcome was the amount of cigarettes smoked per day at the 4-week follow-up (d35). A self-monitoring questionnaire to measure the number of cigarettes smoked during the protocol was asked at baseline, at the end of the stimulation sessions (d5), 2 days later (d7) and at the 4-week follow-up (d35).As secondary outcome measures, each participant completed the following questionaires: (1) Structured Clinical Interview for DSM-IV SCID-I Axis Disorders 1 , were compared between groups and reported in Supplementary Table Baseline characteristics between the two groups (active and sham) were compared for the nominal and ordinal data using a chi-square test (likelihood ratio).treatment is the binary intervention variable  and m1d is the motivation of the subject to stop smoking at baseline. The variable m1d is used in the analysis to strengthen the statistical power in detecting the treatment effect. It also serves to explore the hypothesis of interaction at the baseline between the treatment intervention and the m1d motivation, called here as treatmentXm1d. The covariant m1d is referred to as a moderator and is measured before treatment; this variable/it is not correlated with treatment due to randomization of subjects. The variable m35d is the subject's motivation to stop smoking at d35 of the study. This variable is considered as a mediator in the model. The outcome variable is the average number of cigarettes smoked per day, reported at d35 of the survey, cigar35d. This variable does not violate the temporal order of events between the mediator variable (m35d) and the outcome variable (cigar35d).To analyze how subjects' motivation to quit interacts with the clinical intervention , multivariate linear regressions were used. Mediation analysis is a statistical method used to help answer questions of how a causal agent X conveys its effect on the outcome variable Y  in Equation (1) results:cigar35d is β1 (γ1 + γ3m1di), where m1d moderates the treatment effect. It is observed in the second line that the direct effect of treatment on cigar35d is β2 + β4treat.It can be observed in the first line of Equation (4) that the indirect effect of treatment on As sub-analysis, we performed the same tests dividing participants by smoking frequency: below average, average and above average.Missing data were treated as missing at random (MAR) using the multiple imputation method FIML (full information maximum likelihood) in Mplus software.All statistical analyses were performed using the statistical program Mplus (version 7.4). The syntax of Mplus was obtained from suggestions from session 2.6.5 of Muthén, Muthén and Asparouhov (2016).Out of the 36 participants, 19 were allocated to the active tDCS group and 17 received sham-tDCS. All 36 smokers completed the 5-day tDCS application protocol according to their allocation group . Patients' characteristics and differences between groups (active vs. sham) can be found in Supplementary Table Active and sham groups were homogeneous in relation to age (younger and older than 40 years old), gender, laterality, psychiatric comorbidities , fear of gaining weight, whether they were seriously thinking about quitting smoking, motivation to quit smoking altogether and whether they had quit smoking previously. Fagerström scale questions did not differ between groups either . As the treament variable is dichotomous , the regression can also be interpreted as a difference between the means of cigarette consumption between the two groups; subjects who received active tDCS consumed on average 7.11 cigarettes less than the sham group, when adjusted to these variables. tDCS induced decrease in cigarette consumption was modified by the level of motivation to quit at baseline . However, we observed that the main effect of motivation to quit smoking, recorded both in the baseline (m1d) and in the final phase (m35d), did not influence the average final cigarette consumption alone (cigar35d) . In addition, baseline motivation to stop smoking did not affect motivation at 4-week follow-up (p = 0.108), which did not differ significantly between treatment and placebo groups (p = 0.201). All results are presented in Table In the multivariate analysis (including motivation to stop smoking at d1 and d35), treatment and interaction [motivation (d1) vs. treatment)], we found that the mean number of cigarettes consumed is significantly influenced by the  and d35,average  and higher ] were significant . The treatment was not effective for participants who had below-average cigarette consumption  when compared to placebo.When adding the variable number of cigarettes smoked per day  into the regression, we observed that only the R2 values in Table m35d), 53% of the model variability can be explained by the motivation to stop smoking at baseline (m1d), by the treatment  and by the interaction between both variables (m1d and active tDCS).The Figure In this study, we confirmed our hypothesis that the effect of tDCS on cigarette consumption is partially influenced by motivation to quit. In fact, we found that tDCS effect arises from a conditional analysis of mediation and moderation, where the treatment effect is mediated by the participant's motivation to stop smoking, with both direct and indirect effects. When adding restrictions to the model, we found that the direct effect of the treatment mediated by the participant's motivation to quit appears to be significant only among those who had a cigarette consumption within or above average (>7 cigarettes per day); this significance was not observed among the participants with consumption below average (≤7 cigarettes per day). That is, the treatment did not directly benefit those who smoked less cigarettes than average.Previous tDCS studies aiming at treating smokers targeted the dorsolateral prefrontal cortex (DLPFC), which is thought to be involved in the cognitive control of craving and reward related to smoking  of the DLPFC  . In a recent study assessing the influence of financial motivation on tDCS effects on working memory performance in subjects with low working memory capacity (who did not improve following tDCS), showed that increasing external motivation restored tDCS-related benefits on working memory (Jones et al., As for other conditions, tDCS can induce clinical changes for impaired behaviors. For instance, tDCS can enhance hand motor function when applied over the non-dominant hemisphere but not when the dominant motor cortex is stimulated (Boggio et al., It should be noted that participants were not asked to guess which treatment they received after the stimulation sessions. However, using 30 s of stimulation, which induces the initial itching sensation, is a reliable method of blinding as shown by a previous randomized controlled study using the same stimulation parameters (Fregni et al., Future studies should combine prefrontal tDCS with behavioral treatments to increase motivation to stop smoking or taking an addictive substance, since based on our findings, both tDCS and high motivation to quit are necessary to significantly reduce tobacco consumption. It is now widely accepted that tDCS effects can be manipulated by subject's state (Silvanto et al., All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The handling editor declared a shared affiliation, though no other collaboration, with authors AT and FF. 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.


However, the shallow depth of a light sheet, which is critical for achieving high contrast, well resolved images, adds a significant challenge if fast functional imaging is also required, as multiple images need to be collected across several image planes. Consequently, fast functional imaging of neurons is typically restricted to a small tissue volume where part of the neuronal structure lies within the plane of a single image. Here we describe a method by which fast functional imaging can be achieved across a much larger tissue volume; a custom-built light sheet microscope is presented that includes a synchronized galvo mirror and electrically tunable lens, enabling high speed acquisition of images across a configurable depth. We assess the utility of this technique by acquiring fast functional Ca These dyes can be introduced into the neuron by viral transfection or patch pipette. Classically these dyes have been used with a confocal microscope configured to make a linear scan across a single spine. Fast 2D scanning of a larger area would allow transmission events to be monitored over several dendrites and multiple spines, but 2D confocal scanning is too slow to achieve the 10 or 20 ms scan times required to trace these events over anything but the smallest area. This limitation has two important consequences: 1) Rapid transmission events can be traced only over a very limited set of structures, typically a single spine and the immediately adjacent dendrite. 2) The decision about which spine is of interest must be made before image acquisition begins – interesting events may be occurring at other spines or elsewhere in the dendritic arbour, but will not be captured by the experiment.The neuron is a sophisticated computing device, operating on millisecond time scales to perform complex integrating functions that are not yet fully understood. A principle neuron of the mammalian cortex or hippocampus has an elaborate arbour of long (hundreds of micrometre) processes known as dendrites which are typically covered in tens of thousands of sub-micrometre structures known as spines. Each spine represents the post-synaptic half of a synapse – the intricate structure which mediates the transmission of incoming signals to the neuron. Synaptic transmission causes rapid post-synaptic events such as depolarization of the membrane potential and localized influx of ions such as CaCellular neuroscientists are beginning to turn to light sheet fluorescence microscopy (LSFM) to overcome these limitations. Instead of scanning with a focussed single point of light as in confocal microscopy, LSFM typically uses a cylindrical lens to focus a collimated laser beam in one plane, which when directed through the illumination objective produces a thin sheet of light rather than a point. The detection objective is arranged at right angles to the illumination objective so that its focal plane coincides with the plane of the light sheet. A fast sCMOS  camera enables rapid acquisition of an image from the whole field of view . LSFM wa2+ influx it is necessary to acquire with a temporal resolution of 50-100 volumes/sec. This restricts us to imaging in single-plane mode (XYT), because acquisition of an XYZT stack requires physical movement of the sample stage by a piezoelectric motor, which dramatically slows down the acquisition rate. If we accept this restriction and image in single-plane mode, we find that the meandering path traced by dendrites through a brain slice means that we can only image those short lengths of the dendrite which happen to coincide with the plane of the light sheet  of the detection objective must coincide with the light sheet. One approach to achieve this is to synchronize piezo-driven movement of the detection objective with movement of the light sheet to allow volume imaging without movement of the sample –7. HowevAnother approach is to introduce an electrically tunable lens (ETL) into the detection arm of the microscope, synchronizing movement of the imaging plane with the scanning movement of the light sheet, so that the image formed on the camera's detector is always in focus . This ha2+ events with high temporal resolution. Here we present data from an example experiment which illustrate the promise of fast volume-scanning LSFM for cellular neuroscience.We implement this by synchronizing the movements of the detection plane and the light sheet, while setting the camera's exposure time to equal the period of the scanning cycle bjective . The galbjective . Thus, dThe optical setup is based on the inverted light sheet microscope (LSM) previously reported . BrieflyFast volume scanning is achieved by scanning the illumination light sheet axially with a galvo mirror  on the illumination path. At the same time, an ETL  located immediately behind the detection objective scans the detection plane. Although it is recommended to position the ETL horizontally to minimize optical aberration, we found that placing the ETL at 45 degrees to horizontal has a negligible effect on image quality. Previous studies ,15 utiliThe motions of the detection plane and the illumination light sheet are synchronized so that they always coincide. The trigger output signal from the ETL driver is passed to a function generator  which controls the timing of the scanning galvo mirror. The function generator is operated in burst mode to periodically reset the hardware synchronization between the galvo mirror and the ETL.2) on Millicell culture inserts (Millipore) in culture medium for 2 weeks before use. The culture medium comprised 79% Minimum Essential Media, 20% heat-inactivated horse serum, 1% B-27 (Invitrogen), 30 mM HEPES, 26 mM glucose, 5.8 mM NaHCO3, 1 mM CaCl2, and 2 mM MgSO4. Culture medium was replaced twice weekly. A pyramidal neuron in the CA3 region of the hippocampus was briefly filled with synthetic dyes AF594  and OGB-1  via a patch pipette. After filling, the organotypic slice was transferred to the LSM’s recording chamber (capacity ~4 ml) and perfused at 2 ml/min with carbogenated (95% O2/5% CO2) ACSF. The composition of this ACSF was: (mM) 145 NaCl, 16 NaHCO3, 11 glucose, 2.5 KCl, 1.2 NaH2PO4, 3 CaCl2, and 1 MgCl2, supplemented with 1 mM Trolox to minimize phototoxic damage during imaging. In order to stimulate spiking activity in the slice, about 1 min before imaging the perfusion pump was stopped and a small amount (500 μl) of high-K+ ACSF was added to the perfusion chamber. The composition of this high-K+ ACSF was: (mM) 107.5 NaCl, 40 KCl, 16 NaHCO3, 11 glucose, 1.2 NaH2PO4, 3 CaCl2, and 1 MgCl2.Experiments were conducted in accordance with current United Kingdom (UK) legislation regulating the welfare of animals used for scientific procedures. Organotypic slices were prepared as previously described ,17. In sPrior to the experiment, on the same day, the fast volume-scanning LSM was configured by imaging 500 nm fluorescent beads (Tetraspeck) suspended in agarose. The ETL and galvo mirror were configured to sweep through approximately 20 μm in the Z axis  every 20 ms. The phase difference between the ETL control signal and the galvo mirror control signal was adjusted to optimize image quality .-c.t where a, b, and c are constants and t is time) [2+ events. Candidate events were only accepted if the amplitude of the event  was greater than the estimated noise   to candiLSFM see .Post hoc analysis of the image data reveals details of localized, rapid Ca2+ influx events occurring at various locations and their spread or otherwise through the dendritic arbour. This wealth of information cannot be acquired by a classical confocal line scan approach, which by its nature requires the investigator to decide ahead of time upon a single location of interest.To demonstrate the potential of our approach, we present data acquired from a single imaging experiment, where the dendritic arbour of a living pyramidal neuron was scanned for 10 seconds using fast volume-scanning LSFM. 2+-sensitive dye Oregon Green BAPTA-1 (OGB-1), along with Alexa Fluor 594 (AF594) for structural imaging, then transferred the slice to the LSM. About 1 minute before the start of image acquisition, a small amount of high-K+ ACSF was added to the recording chamber to stimulate spiking activity in the hippocampal slice. A portion of the filled neuron’s basal dendritic arbour was imaged using the fast volume-scanning technique described above  e at about the 2.8 sec time point. A short distance (less than 5 μm) along the dendrite in either direction (ROIs f and d) the same event is apparent but with a slower rise time and a much reduced amplitude. Further (about 10 μm) towards the soma the first event is not visible, while the second event is still visible though reduced in amplitude and delayed (ROI g). Further away from ROI e (ROIs c and h) or in another dendrite (ROIs a and b) there is no sign of the event, showing that this cannot be interpreted as a back-propagating action potential originating from the soma, as this would be visible throughout the dendritic arbour. It is likely that this segment of the recording shows a strong synaptic transmission event (or sequence of two events) occurring at one or two synapses in the vicinity of ROI e. The resulting Ca2+ influx is seen propagating a short distance along the dendrite on either side of the event’s origin, possibly reflecting calcium-induced calcium release (CICR) [Using a patch electrode, we filled a pyramidal neuron in the CA3 region of a cultured rat hippocampal slice with Caed above . The imae (CICR) , or evene (CICR) , but it 2+ transient event starting at about the 9.3 sec time point, which seems to have the sharpest rise time and largest amplitude at ROI c, but is also visible at least 50 μm away in either direction along the dendrite , but not on a different dendrite (ROIs f and g), which again rules out a back-propagating action potential as an interpretation. Note that at ROI e there is another, earlier Ca2+ transient event at about the 8.6 sec time point which in contrast does not propagate very far along the dendrite. A likely interpretation is that we have two synaptic transmission events: one at a synapse in the vicinity of ROI c which engages some kind of amplifying mechanism such as CICR or dendritic spiking to enable the Ca2+ influx to propagate at least 50 μm along the dendrite; and another transmission event at a synapse in the vicinity of ROI e which fails to propagate a Ca2+ influx along the dendrite.In Fig. 3+ ACSF. Comparison with 2+ transient event is visible at about the 1.3 sec time point at ROI d. This event does not appear to propagate far along the dendrite, but it is hard to make a firm interpretation because of the fragmentary nature of the in-focus dendrites available in this single-plane image stack.For comparison a second 10 sec image acquisition was performed on the same neuron, but this time the LSM was operated in single-plane mode  This demonstrates that our custom-built fast volume-scanning LSM is also capable of acquiring structural images with spine-level resolution.We also show details of a structural image stack acquired at the close of the same experiment  Fast volume-scanning LSFM and single-plane LSFM are optimized for different tasks. Single-plane LSFM is best suited to an experiment which is interested in fast functional imaging in a restricted length of dendrite of the order of 20 μm long, where perhaps a single synapse might be manipulated using a technique such as glutamate uncaging, and the functional effect of this on a handful of nearby spines monitored with high temporal resolution. It is relatively simple to find such a short length of contiguous dendrite that is in the plane of the light sheet. Fast volume-scanning LSFM on the other hand is best suited to an experiment that is designed to collect fast functional imaging data over a wider area of the dendritic arbour, covering multiple dendrites and multiple spines in a single imaging scan. Note that the depth of the light sheet’s scan in the Z axis, which in our example experiment was configured at 20 μm, can be configured to any value consistent with the operational ranges of the signals driving the galvo mirror and the ETL.In this report we have presented imaging data acquired by a variant of our previously reported neuroscience-optimized LSM  modifiedht sheet ,15. HoweWe conclude that fast volume-scanning LSFM holds considerable promise for studies in cellular neuroscience. It has the potential to acquire fast functional imaging data across wide areas of a neuron’s dendrites. This raises the prospect of understanding in more detail than ever before the way that synaptic transmission events interact with events at neighbouring or more distant synapses, and are integrated and propagated (or suppressed) through various compartments and branches of the dendritic arbour.


Gastrointestinal illness (GI) has been associated with heavy rainfall. Storm events and periods of heavy rainfall and runoff can result in increased microbiological contaminants in raw water. Surface water supplies are open to the environment and runoff can directly influence the presence of contaminants. A time-stratified bi-directional case-crossover study design was used to estimate associations of heavy rainfall and hospitalizations for GI. Cases of GI were identified as in-patient hospitalization with a primary diagnosis of infectious disease associated diarrhea [ICD-9 codes: specified gastrointestinal infections 001–009.9 or diarrhea 787.91] among the residents of New Jersey from 2009 to 2013 resulting in a final sample size of 47,527 cases. Two control days were selected on the same days of the week as the case day, within fixed 21-day strata. Conditional logistic regression was used to estimate odds ratios controlling for temperature and humidity. To determine potential effect modification estimates were stratified by season (warm or cold) and drinking water source . Stratified analyses by drinking water source and season identified positive associations of rainfall and GI hospitalizations in surface water systems during the warm season with no lag  and a 2-day lag . Positive associations in ‘Other’ water source areas  during the warm season with a 4-day lag were also found. However, there were no statistically significant positive associations in groundwater systems during the warm season. The results suggest that water systems with surface water sources can play an important role in preventing GI hospitalizations during and immediately following heavy rainfall. Regulators should work with water system providers to develop system specific prevention techniques to limit the impact of heavy rainfall on public health. Heavy rainfall has been associated with outbreaks of waterborne diseases –3, and sUnlike surface water, groundwater is thought to be naturally protected from increases in turbidity and microbial contamination from rainfall and runoff and therefore does not require disinfection plus filtration for particle removal . HoweverSince severe weather events are predicted to occur more frequently and with greater severity in the future , a more This research involves human participants’ personal health information and was approved by the New Jersey Department of Health (NJDOH) Institutional Review Board (IRB) for human subjects research and later transferred to Rowan University IRB as the IRB of record for this study. Informed consent for this work was waived due to practicability and determination of minimal risk.The study population comprised residents of the State of New Jersey during 2009 through 2013. Cases of GI among the population were identified as in-patient hospitalizations with a primary diagnosis of infectious disease associated diarrhea [ICD-9 codes: specified gastrointestinal infections 001–009.9 or diarrhea 787.91] in the New Jersey Hospital Discharge Data Collection System.We employed a time-stratified bi-directional case-crossover study design, which compares each case’s exposure to referent (control) days. Since each case serves as his or her own control, individual-level confounding factors that remain constant over a short period of time  are controlled for.The date of hospital admission served as the case day. Two control days were selected from those days on the same day of the week as the case, and that were within the same, fixed 21-day stratum as the case . Further, bi-directional control sampling allows theses control days to be selected from weeks before and/or weeks after the case day . SelectiGlobal Surface Summary of the Day data, which includes daily measures of rainfall, temperature, and relative humidity for 26 stations in New Jersey, were obtained from the National Centers for Environmental Information . Each ofThe New Jersey Department of Environmental Protection (NJDEP) developed a geographic information system (GIS) layer defining the boundaries of community water system (CWS) in the state . Each ofUsing the 2010 U.S. Census Bureau, the percentage of persons living in poverty by census tract was used to define socioeconomic status (SES) . A high Each GI case was geocoded using the residential street address appearing in the hospitalization record, with 94% completed matching. Cases that could not be geocoded to residential address were excluded. Cases were spatially linked to one of the five identified climate regions in New Jersey. Each case-day and control-day was assigned meteorological exposures based on day and the assigned climate region. Each case was also spatially linked to a New Jersey CWS or ‘Other’ water source area, and to a census tract.th percentile (0.37 inches) for same day rainfall or (0.33 inches) for 3-day average rainfall among control days. Previous research has indicated that associations with precipitation and outbreaks of disease occur at or above the 90th percentile [Conditional logistic regression was used to estimate the relative odds of exposure to rainfall events, comparing case days to control days. Odds ratios (OR) and 95% confidence intervals (CI) were computed. Rainfall was categorized as ‘heavy rainfall’ if it was equal to or greater than the 90rcentile , 10. AllMicrobiologically-associated GI hospitalizations could have latency periods that include the time from a rainfall event to exposure of an individual at the tap, the time from exposure to symptom onset, and the time from symptom onset to hospitalization. Therefore, exposure lagging of up to 14 days was explored. To explore the potential for cumulative impact of rainfall events, 3-day average rainfall was computed with 3-day, 5-day, and 7-day lags.th percentile was compared to each increasing 5th percentile up to the 90th percentile for each water source. ORs and 95% CIs were graphed to explore the shape of the dose-response relationship.Stratification by season defined as warm  or cold (November through March) was performed to assess seasonal effect modification by season. Models were also stratified by water source: groundwater, surface water, and ‘Other’ to assess effect modification by water source. Finally, models were stratified by gender, age group  since the pediatric and older populations may be more susceptible to GI events, race , and census tract-based SES. Based on findings, a threshold analysis was conducted in which the bottom 50A total of 47,527 cases of hospitalization for GI were identified, and resulted in the selection of 95,054 control days. The service population of CWS across the state varies widely. Therefore, a large proportion of cases were linked with larger CWSs. One CWS served by a surface water source served approximately 11% of cases, and supports over 790,000 people (about 9% of New Jersey’s population). Six other systems each served about 5% of the cases. The ‘Other’ category consisting of areas served by very small CWS, private wells, or unknown was 15% of the study population, which compares to the current estimate of 12% of the New Jersey population served by private wells.Cases were more often female (59.0%) than male (41.0%). A larger proportion of cases were White (67.3%) than Black (20.5%) or other race group. About 5% of cases were under age 5 years, and 43% were age 65 years or over. Approximately 18% of cases lived in census tracts categorized for this analysis as low SES. Demographic characteristics of cases differed by drinking water source . From suAdjusted ORs and 95% CIs of GI hospitalization and heavy rainfall are presented for various lag periods for the full year and by season in For the full year, there was an inverse association of GI with rainfall with a 3-day lag  and a positive association of GI with 3-day average rainfall with a 7-day lag . During the warm season, positive associations of GI hospitalization and heavy rainfall were seen with no lag , a 2-day lag , and a 4-day lag . During the cold season there were statistically significant inverse associations for same-day lag through 3-day lag, and for 3-day averages with no lag and a 3-day lag.For surface water sources, GI was positively, statistically significantly associated with heavy rainfall during the warm season on the same day and with a 2-day lag . During the cold season, associations for surface water sources were inversely, statistically significantly associated with same-day through 3-day lag periods as well as with a 3-day average precipitation with no lag and with a 3-day lag. For ‘Other’ water sources, there was a statistically significant positive association with a 4-day lag period  during the warm season, and statistically significant association with a 3-day average with a 5-day lag  during the cold season.Adjusted ORs and 95% CI of associations by age group, White and Black race, and SES categories for surface water systems during the warm season are presented in th percentile to each increasing 5th percentile of rainfall levels during the warm season for both same day and 4-day lag for all water sources combined and for each water source type  produced an increased effect  compared to 90th percentile to all other rainfall days presented in th percentile and the 90th percentile are likely to have strengthen the association. An increase in effect estimate for GI hospitalization with increasing rainfall may also be indicated in ‘Other’ water source type for a 4-day lag, although no associations are statistically significant. There were no statistically significant associations for groundwater at any percentile for either same day or 4-day lagThreshold analysis compared the bottom 50rce type . The anaGastrointestinal illness (GI) attributable to drinking water presents a large nationwide burden each year, with estimates of 4.3 to 11.7 million cases per year  and 16.4Studies have shown mixed results regarding associations of precipitation and waterborne disease. A review of studies of extreme precipitation and drinking water-related waterborne infections identified 11 studies with positive associations, six with no associations, and three with mixed results . StudiesThere is evidence that indicators of microbiological drinking water quality parameters are associated with increased waterborne disease risk, and that risk may be modified by source water type. Excess risk of emergency department visits for diarrhea and source water turbidity in New York City peaked at a 6-day lag  and a stIn our study, drinking water source modified associations of rainfall and illness in which positive associations were limited to surface water and ‘Other’ sources of drinking water, but not groundwater. Threshold analyses presented in We hypothesized associations to be lagged due to environmental transport, disease latency, and health-seeking behaviors. Curriero et al. 2001) found a 51% increase of waterborne disease outbreaks with a 2-month lag and a time-series analysis among two Inuit communities in Canada from 2005–2008 found significantly positive associations of infectious gastrointestinal illness with high 2-week and 4-week lagged water volume input  [ found a Our observed associations of little to no lag in surface water does not account for the lag time that would be associated with a hypothesized delay for a mechanism of event to exposure to disease. It is possible that there are vulnerabilities in some surface water systems which could drive these acute positive associations following rainfall, including open-air reservoirs and water storage tank susceptibilities, but future research is required to investigate this. A large time-series analysis in Wisconsin found positive associations with rainfall and GI with a 4-day lag [Stratification by season was necessary to detect associations with rainfall and hospitalization with GI , 41. SeaOur study found that children under 5 years of age were at an increased relative risk of GI hospitalizations following heavy rainfall events than other age groups, and some evidence of increased relative risk among those residing in census tracts in the lowest SES category in comparison to higher categories. Race did not modify effect estimates. Teschke et al. (2010) found an elevated odds ratio for hospitalizations for intestinal illness among children aged 1 to 4  and also individuals in the lowest two household income quintiles at an increased risk of intestinal infections  . Our thrNon-differential exposure misclassification may have introduced bias towards the null. Meteorological data were averaged from various stations over each of the five climate regions. This allowed for complete meteorological information but daily assigned rainfall exposure may not accurately reflect the actual conditions that may have impacted water quality where a particular case resides. Exposure assignment was based on residential address, but actual exposure to drinking water on any given day may have occurred elsewhere such as at work or school. Assignment of source water may have been inaccurate since some water systems, especially larger ones, are particularly complex, often utilizing both surface and groundwater in different parts of the service area and at different times throughout the year. More refined understanding of water distribution through time within complex CWSs would be needed to better classify the drinking water source for each case. The ‘Other’ category is not a homogeneous grouping regarding water quality, vulnerability to heavy precipitation, or source water type.Although the U.S. government is responsible for implementing the Safe Drinking Water Act, each state has the authority to create more stringent and/or additional standards or rules for water systems in their jurisdiction. For example, all CWS in New Jersey are required to maintain a detectable level of disinfectant regardless of whether source water comes from a groundwater or surface water source, unlike the non-disinfected municipal water systems studied by Ueijo et al. (2014). We believe it is important to understand the role drinking water plays in endemic rates of GI especially as weather factors and events are predicted to have larger impacts in the future. However, the role of water systems in associations of precipitation and disease will likely vary based on the drinking water regulations and source waters particular to each place. Therefore, more studies in more geographic areas will be needed to understand the specific and unique vulnerabilities of water systems with varying source characteristics and regulatory environments.Findings from this project have identified needs for further research. Additionally, more work has to be done to identify surface water system vulnerabilities. This may allow us to pinpoint the driving factors or identify water systems that may be driving observed positive associations. Future research in New Jersey may also include performing time-series analyses or analyses restricted to one large CWS served by a surface water source. This would allow a more detailed exposure assessment and may allow the linkage of rainfall, snowfall, melt events, and other possible modulating water quality parameters to GI.Water systems with surface water sources can play an important role in preventing GI hospitalizations during and immediately following heavy rainfall. Although more research is required to determine factors that may explain the mechanism of risk or to explain a mechanism unrelated to drinking water, surface water systems may consider additional protection measures during heavy rainfall events such as source water protection. Regulators should work with water system providers to develop system specific prevention techniques to limit the impact of heavy rainfall on public health.S1 Table(DOCX)Click here for additional data file.


Temporal resolution needed for Japanese speech communication was measured. A new experimental paradigm that can reflect the spectro-temporal resolution necessary for healthy listeners to perceive speech is introduced. As a first step, we report listeners' intelligibility scores of Japanese speech with a systematically degraded temporal resolution, so-called “mosaic speech”: speech mosaicized in the coordinates of time and frequency. The results of two experiments show that mosaic speech cut into short static segments was almost perfectly intelligible with a temporal resolution of 40 ms or finer. Intelligibility dropped for a temporal resolution of 80 ms, but was still around 50%-correct level. The data are in line with previous results showing that speech signals separated into short temporal segments of <100 ms can be remarkably robust in terms of linguistic-content perception against drastic manipulations in each segment, such as partial signal omission or temporal reversal. The human perceptual system thus can extract meaning from unexpectedly rough temporal information in speech. The process resembles that of the visual system stringing together static movie frames of ~40 ms into vivid motion. Speech can remain considerably intelligible even when it is drastically manipulated in the temporal domain. One example is the word intelligibility (articulation) of speech in which 50-ms portions are alternately played and silenced, as reported by Miller and Licklider , who disNeuroscientific research performed in the last decade has suggested that different types of neural oscillations are involved in the segmentation and organization of speech into perceptual units  makes it essentially impossible to control both time and frequency very accurately , we used not only mosaic speech, but also locally time-reversed speech with smoothing and without smoothing, as well as the original speech. Locally time-reversed speech was made by reversing each segment in time as in Figure n = 4 in Experiment 1 and n = 20 in Experiment 2) were asked to write down what they heard in Japanese hiragana letters, each corresponding clearly in most cases to one mora—basic phonological units of Japanese, which are in many cases equal to and sometimes shorter than syllables. The percentage of correct mora identification was calculated by counting the number of written morae that corresponded to the morae pronounced in the three sentences assigned to each stimulus condition . Multiple comparisons after that (following Siegel and Castellan) indicated significant differences between mosaic speech and locally time-reversed speech with/without rise and fall times . The difference between the two types of locally time-reversed speech was not significant. In sum, the participants' performance was significantly better for mosaic speech than for locally time-reversed speech, and the 50% threshold for Japanese mosaic speech exceeded 70 ms.A Friedman two-way analysis of variance by ranks , can be conveyed by 3–4 still  frames. It thus is possible to synthesize intelligible speech connecting still frames in time, as if they were Lego blocks.In order to understand the mechanism of speech communication, it is of vital importance to determine how far the speech signal can be degraded in the temporal dimension. It should be one of the unavoidable steps to examine how long homogeneous temporal units can be, still conveying linguistic information. To employ locally time-reversed speech systematically has been a substitute for this paradigm  or 20 (Experiment 2) native-Japanese speakers participated. They were 4 men of 21–25 years old (Experiment 1) or 9 women and 11 men of 19–25 years old (Experiment 2), all with normal hearing.Four stimulus types were generated: (1) original speech with a 5-ms rise and a 5-ms fall time Figure , (2) mosJapanese speech samples were obtained from the “NTT-AT Multi-Lingual Speech Database 2002.” The samples were spoken by a female native-Japanese speaker, and with a sampling rate of 16 kHz with 16-bit quantization. The samples were edited to remove irrelevant silent portions and noises, and converted into computer-oriented audio (.wav) files with a sampling frequency of 22.05 kHz using Praat  for each block. The participant was first asked to perform a practice block consisting of 20 trials. The sentences used in the practice block and the warm-up trials were not used again.The research was conducted with prior approval of the Ethics Committee of Kyushu University; all methods employed were in accordance with the guidelines provided by the Japanese Psychological Association. The participants provided written informed consent prior to their participation.The stimuli were presented to the participant in a soundproof room, from a computer (Frontier KZFM71/N) with an audio board (E-MU 0404) that was installed outside the soundproof room. From the computer, the stimuli were passed through an audio processor (Onkyo SE-U55GX), a low-pass filter , a graphic equalizer (Roland RDQ-2031), and a headphone amplifier (STAX SRM-3235), before being presented to the participant through headphones (STAX SR-307). The equalizer was used to keep the flat shape of the frequency characteristics of the headphones, while the low-pass filter was used for anti-aliasing. Since the sampling frequency of the speech files was 22.05 kHz, the cut-off frequency of 15 kHz could not suitably deal with aliasing in between 14.05 and 15 kHz. The sound energy related to this range, however, was negligible. The audio output level was calibrated so that a 1-kHz pure tone of the same intensity as the average intensity of the stimuli was at 70 dBA with a precision sound level meter (Naganokeiki 2071) mounted with an artificial ear (Brüel and Kjær 4153).YN: designed the study, wrote prototypes of computer programs, analyzed the data, and wrote the paper; MM: designed the study, and collected and analyzed the data; KU and GR: prepared for the experiment, analyzed the data, and wrote the paper.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.


The aim of the study was to compare the clinical outcomes following elective and traumatic total hip arthroplasty in Parkinson's disease patients. Ten patients with osteoarthritis comprise the elective group . Thirteen patients with femoral fracture comprise the hip fracture group . All patients were followed up at 6 and 36 months postoperatively and at the time of the latest follow-up. Despite the significant improvement in Merle d'Aubigné-Postel and pain scores, disability related to Parkinson's disease increased during the follow-up. Whereas more than 1/3 of hip fracture patients and all elective patients walked independently at 36 months after total hip arthroplasty, 43% of living patients from both groups were able to walk independently at the time of the latest follow-up. The medical complications were seen mainly in patients with hip fracture. Excellent pain relief with preserved walking ability without support of another person and acceptable complication profile was observed in Parkinson's disease patients at 36 months after elective total hip arthroplasty. This procedure may be indicated in Parkinson's disease patients after careful and individualized planning. The prevalence of Parkinson's disease and the incidence of hip fractures mirror an ageing population living longer , 2. ParkThe aim of the study was to compare the short- to mid-term clinical outcomes following elective and traumatic total hip arthroplasty in patients with Parkinson's disease focusing on the assessment of risks and benefits of surgery.All patients with a confirmed diagnosis of Parkinson's disease having total hip arthroplasty at our institution between January 2005 and December 2012 were enrolled in a retrospective analysis. In total, 24 total hip arthroplasties were implanted in 23 patients, 8 men (35%) and 15 women (65%). The primary indication for surgery was osteoarthritis for 10 hips; these 10 patients comprise the “elective group” with a mean age at operation 74 years (65 to 82). Thirteen patients underwent total hip arthroplasty for proximal femoral fracture (one patient had hip fracture subsequently on both sides); they comprise the “hip fracture group” with a mean age at operation 76 years (67 to 83) . The resAll the procedures were performed via a standard anterolateral Watson-Jones approach to the hip joint. In order to maximize sample size in the elective and hip fracture groups, implant design was not a controlled variable. The details of components with regard to stability of the implanted nonconstrained total hip arthroplasty are depicted in Maintained independent ability to walk: being able to walk without support from another person (with aids if necessary).Not maintained: support from another person or use of a wheelchair required.Outcome measure analysed in the study included Charnley's modified Merle d'Aubigné and Postel scoring system . As a reComplications were recorded throughout the follow-up period. All patients were followed up prospectively before surgery, at 6 months and 36 months postoperatively, and at the time of the latest follow-up.In the elective group consisting of 10 hips, the mean follow-up was 82 (33–143) months. One patient in the elective group died at 33 months postoperatively from causes unrelated to the surgery (pulmonary tumor). For 14 hips in the hip fracture group, the mean follow-up was 54 (1–143) months. Five patients in the hip fracture group died during follow-up at 1, 3, 4, 11, and 30 months postoperatively.https://www.ncss.com). Since the type of data of all the tested parameters is ordinal, we opted for using the Wilcoxon signed-rank test. A value of P < 0.05 was considered to be significant. For the purpose of comparison of the elective with the hip fracture group, only the data of the patients who survived until the last follow-up (median 82 months for the hip fracture and 72 months for the elective group) were used.Measurement data were processed and statistically evaluated with the help of MS Excel 2013  and NCSS 2007 . NCSS 2007. NCSS LLC, Kaysville, Utah, USA. P < 0.001) for both groups; there was no difference between the 6 months and 36 months and between the 36 months and latest follow-up Merle d'Aubigné and Postel scores in the elective group and in the hip fracture group too.The statistically significant difference between the medians of Merle d'Aubigné and Postel score in the elective and hip fracture group was recorded preoperatively, at 6 and 36 months after total hip arthroplasty. There was no statistically significant difference between the two groups during the last follow-up . The impP < 0.001) for both groups with no differences between the 6 months and 36 months pain scores and between the 36 months and latest follow-up scores in the elective group and in the hip fracture group too. Of the 20 patients (21 hips) of both groups followed up at 6 months, 18 hips had no pain and 3 hips had only slight pain. Of the 17 patients (18 hips) of both groups followed up at 36 months, 17 hips had no pain and 1 hip had only slight pain. There was no pain observed in 18 hips of both groups at the time of latest follow-up.The elective group had preoperatively significantly higher pain score when compared with the hip fracture group. There was no statistically significant difference between the two groups during the 6 months, 36 months, and the last follow-up . The impDespite the improvement in Merle d'Aubigné and Postel and pain scores, disability related to Parkinson's disease increased during the follow-up; neurological progression was noted in 83% of all our patients. At the time of latest follow-up 57% of all the patients had progressed to functional stage IV or V .All patients in both groups were able to walk independently before surgery and functional status deteriorated over time as seen in The complications are listed in Four patients in the hip fracture group died during first year after surgery: three of pneumonia and one of cerebrovascular accident. The overall 1-year mortality for the hip fracture group was 28.6%.Total hip arthroplasty is one of the major successes of modern medicine . As a reOur study demonstrates a clear improvement in hip pain following total hip arthroplasty in patients with Parkinson's disease as supported by improved Merle d'Aubigné and Postel and pain scores. This improvement was maintained from 6 months after surgery to the latest follow-up in the elective and hip fracture group. Although the functional status of Parkinson's disease patients deteriorated over time, we observed that patients in the elective group benefited from excellent pain relief and were able to walk without support of another person at 36 months after total hip arthroplasty which was indicated for osteoarthritis with severe hip joint pain preventing activities of daily living. Ten of living patients from both groups (43%) were still able to walk without support of another person at the time of the latest follow-up.Although variable mortality following hip fracture in Parkinson's disease patients has been reported, we found it comparable with 1-year mortality published in non-Parkinson's disease patients , 17. HowObstructive respiratory pattern due to neuromuscular dysfunction predisposes to retained secretion, atelectasis, and pulmonary infection . We founThe musculoskeletal manifestation of Parkinson's disease include tremor, rigidity, contractures, bradykinesia, dystonia, and postural instability which theoretically predispose patients to dislocation of the hip . HistoriRecently Parkinson's disease patients had an approximately twofold risk of hip dislocation . AdductiDespite early mobilization started on the first postoperative day, intensive postoperative rehabilitation, and patient's care, the dislocation rate of nonconstrained total hip arthroplasty in our study (8.3%) is comparable with recently reported rate in nationwide registry-based case-controlled study (6.1%) . Both arMost Parkinson's disease patients experience falls as a result of disease symptoms and many have recurrent episodes . It was With increasing prevalence of Parkinson's disease, falls and fractures are anticipated to have a major impact on health care systems in the coming decades . SeveritThis retrospective study is limited by the small patient numbers and short duration of follow-up. Nevertheless the follow-up was sufficient to reveal a clinical outcome following elective and traumatic total hip arthroplasty in patients with Parkinson's disease. Total hip arthroplasty is a viable alternative for these patients if the surgery is individualized and carefully planned. A multidisciplinary team comprised of health professionals, including the neurologist, geriatrician, and physiotherapist, should be involved to maximize patient outcome . Early dIn conclusion, total hip arthroplasty in patients with Parkinson's disease is challenging due to higher risk of medical complications which were seen mainly in patients with hip fracture. Excellent pain relief with preserved walking ability without support of another person and acceptable complication profile was observed in Parkinson's disease patients at 36 months after elective total hip arthroplasty. Elective total hip arthroplasty may be indicated in patients with Parkinson's disease after careful and individualized planning.