Motor sequences are formed through the serial execution of different movements, but how nervous systems implement this process remains largely unknown. We determined the organizational principles governing how dirty fruit flies groom their bodies with sequential movements. Using genetically targeted activation of neural subsets, we drove distinct motor programs that clean individual body parts. This enabled competition experiments revealing that the motor programs are organized into a suppression hierarchy; motor programs that occur first suppress those that occur later. Cleaning one body part reduces the sensory drive to its motor program, which relieves suppression of the next movement, allowing the grooming sequence to progress down the hierarchy. A model featuring independently evoked cleaning movements activated in parallel, but selected serially through hierarchical suppression, was successful in reproducing the grooming sequence. This provides the first example of an innate motor sequence implemented by the prevailing model for generating human action sequences.DOI:http://dx.doi.org/10.7554/eLife.02951.001 Anyone who has ever lived with a cat is familiar with its grooming behavior. This innate behavior follows a particular sequence as the cat methodically cleans its body parts one-by-one. Many animals also have grooming habits, even insects such as fruit flies. The fact that grooming sequences are seen across such different species suggests that this behavior is important for survival. Nevertheless, how the brain organizes grooming sequences, or other behaviors that involve a sequence of tasks, is not well understood.Fruit flies make a good model for studying grooming behavior for a couple of reasons. First, they are fastidious cleaners. When coated with dust they will faithfully carry out a series of cleaning tasks to clean each body part. Second, there are many genetic tools and techniques that researchers can use to manipulate the fruit flies' behaviors. One technique allows specific brain cells to be targeted and activated to trigger particular behaviors.Seeds et al. used these sophisticated techniques, computer modeling, and behavioral observations to uncover how the brains of fruit flies orchestrate a grooming sequence. Dust-covered flies follow a predictable sequence of cleaning tasks: beginning by using their front legs to clean their eyes, they then clean their antennae and head. This likely helps to protect their sensory organs. Next, they move on to the abdomen, possibly to ensure that dust doesn't interfere with their ability to breathe. Wings and thorax follow last. Periodically, the flies stop to rub their legs together to remove any accumulated dust before resuming the cleaning sequence.Seeds et al. activated different sets of brain cells one-by-one to see if they could trigger a particular grooming task and found that individual cleaning tasks could be triggered, in the absence of dust, by stimulating a specific group of brain cells. This suggests each cleaning task is a discrete behavior controlled by a subset of cells. Then Seeds et al. tried to stimulate more than one cleaning behavior at a time; they discovered that wing-cleaning suppressed thorax-cleaning, abdomen-cleaning suppressed both of these, and head-cleaning suppressed all the others. This suggests that a ‘hierarchy’ exists in the brain that exactly matches the sequence that flies normally follow as they clean their body parts.By learning more about how the brain coordinates grooming sequences, the findings of Seeds et al. may also provide insights into other behaviors that involve a sequence of tasks, such as nest building in animals or typing in humans. Following on from this work, one of the next challenges will be to see if such behaviors also use a ‘suppression hierarchy’ to ensure that individual tasks are carried out in the right order.DOI:http://dx.doi.org/10.7554/eLife.02951.002 Animals engage in a variety of complex serial behaviors that are essential for survival, such as nest building, communication, courtship, and prey capture. Serial behaviors can be subdivided into distinct motor actions that are mutually exclusive and often proceed in a given sequence . While oThere are two main hypotheses about mechanisms that drive the sequential progression of actions during serial behavior. One hypothesis suggests that each action in a series triggers performance of the next action . This isDrosophila melanogaster permits subdivisions of behavior to be identified and manipulated in freely moving flies by expressing neural activators in subsets of neurons in the brain. This approach has revealed that acute neural activation can trigger specific behaviors and subcomponents of behaviors, such as abdominal bending and courtship song (two subcomponents of the courtship sequence)  . While tDrosophila grooming is well described by a parallel model of serial behavior.Here, we study the organizational principles underlying the neural control of serial action selection in an innate behavior common to most limbed animals—grooming . GroominWe tested whether flies would prioritize cleaning of specific body parts when they were completely dirty. This was achieved by coating flies in dust, paired with measurements of changing dust distributions on the body surface  and manuUAS-dTrpA1) . This suggests that the targeted neurons are sufficient but not necessary for driving their respective cleaning movements. All lines tested could activate continuous cleaning of their corresponding body parts for long periods of time (at least 25 min). Thus, grooming can be decomposed into specific motor programs (hereafter referred to as cleaning modules) that can be independently activated by subsets of neurons. Experimental induction of these cleaning modules via GAL4-expressed dTrpA1 allowed us to directly test how they are sequentially executed during the progression of grooming.The modularity of grooming is evident from observations that individual cleaning movements can be evoked by locally applied stimuli . This le-dTrpA1)  in diffe-dTrpA1)  and scre-dTrpA1) , R26B12.We postulated that coating flies with dust leads to the simultaneous stimulation of all the cleaning modules, but because of body mechanical constraints, these competing modules are mutually exclusive and therefore must be performed one at a time. Such temporal conflicts between competing behaviors can be resolved if the performance of one behavior suppresses the others, such that only one is selected at a time . BuildinOur GAL4 lines allowed us to test this hierarchical suppression hypothesis by artificially driving cleaning of specific body parts while simultaneously dusting the flies to induce competing cleaning movements. For this experiment, we selected lines that could induce a particular cleaning module and lacked additional phenotypes that might confound our grooming-specific interpretations . Figure Although the priority order of cleaning module execution can be attributed to a suppression hierarchy, the sequential progression of grooming requires the termination of the hierarchically superior cleaning module followed by selection of the next module. One possible explanation for the observed progression is that each module sequentially triggers the next module in the series, analogous to an activation chain . We testUAS-dTrpA1 flies shown in An alternative way by which grooming could progress is through a combination of the acute sensory cues provided by dust and the intrinsic suppression hierarchy among the cleaning modules. To test this hypothesis, we took advantage of the fact that artificial activation of specific cleaning modules blocks grooming of subordinate body parts . Thus, i, the removal of dust by a module is represented with blunt arrows from the winner-take-all layer to the dust). Grooming proceeds from the module with the highest activation level to that with the lowest until dust is removed from all of the body parts. This organization leads to the emergence of sequential cleaning of body parts.We found that hierarchical suppression can mediate the selection of one among many simultaneously stimulated cleaning modules that are in competition for output. Between the proposed models describing how nervous systems execute serial actions, hierarchical suppression through competition is a core feature of parallel response models rather than activation chain models . The genFigure 4AThe general strategy of hierarchical suppression can be implemented using two related schemata. We simulated both possible implementations with a computational model to test if they were sufficient to explain the progression of grooming that we observed. The winner-take-all layer is the same for both implementations. Winner-take-all neural networks can be modeled using all-to-all inhibitory connections between the different units; however, conceptually, they simply select the most active unit and inhibit the rest . In the The model of hierarchical suppression also replicates the returns to previous cleaning modules that we observed with dusted flies . Based oIn addition to simulating the grooming progression, the model recapitulates two other important features of our experimental data. First, it can mimic the constitutive cleaning phenotypes of GAL4 lines expressing dTrpA1 in the absence of dust. Constitutive activation was implemented by setting a particular body part to be fully dusted regardless of the amount of cleaning done (as if the sensory neurons were constantly active). This resulted in the continual cleaning of a particular body part without triggering any other cleaning modules . Second,Our model explains how the sequential selection of cleaning modules occurs, but could it also explain the frequent alternations between cleaning the body parts and leg rubbing that occur throughout the grooming progression? These alternations are an important and prominent feature in normal grooming behavior for efficient dust removal . In ordeOur model predicts that leg rubbing is among the most hierarchically superior of the cleaning modules. Therefore, if leg rubbing is constitutively activated, it should suppress cleaning of the other body parts in a competition experiment where flies are coated in dust. We tested this prediction by using a GAL4 line that drove strong leg rubbing of both the front and hind legs , R17F11.Hierarchical associations among different available behavioral options allow for those behaviors with the highest value for a given circumstance to be selected while all others are suppressed. These concepts are often referred to as behavioral choice, action selection, motor program selection, or decision making . For exaA significant finding of this work is the experimental demonstration that hierarchical suppression among many behaviors leads to the emergence of a sequence. Although much attention has focused on the role of hierarchical suppression in selecting between behavioral choices, there is little experimental evidence to support its role as a mechanism for eliciting action sequences. The grooming sequence may be considered as a series of ‘forced choices’ among different competing cleaning modules that are selected in priority order through suppression. As each body part is cleaned, suppression of the other cleaning modules is lifted, and a new round of competition between the remaining cleaning options occurs. Our work lends strong support to an emerging view that hierarchical suppression is a ubiquitous action selection mechanism, whether it is used in deciding among available behavioral choices, or selecting movements performed in complex serial tasks. Thus, the evolutionarily ancient ability to select among competing behaviors through hierarchical suppression may have provided the necessary infrastructure to perform motor actions in sequence .Activation chain and parallel models describe two different mechanisms for generating sequential behavior. Activation chains  are the simplest form of sequential execution. These work through feedforward activation of the next movement by the previous one . ParalleTwo lines of evidence argue against a primary role for an activation chain in driving the dust-induced grooming sequence. First, each cleaning movement can be activated by local stimulation of its respective body part without triggering other movements . Either We find that the grooming sequence has features that are similar to parallel models of serial behavior. Both involve the parallel activation of premotor units in the sequence. The activation of the cleaning modules occurs through dust sensing, where each module is independently stimulated by a dust stimulus on its corresponding body part. Coating the body of the flies in dust leads to the parallel stimulation of the modules, and thus competition among them. Parallel models resolve such simultaneous activation schemes by creating a gradient of activity levels among the units that are executed in the sequence and selecting the one with the highest activity in a winner-take-all network. Our computational model of grooming works through a similar mechanism . We findThe finding that grooming behavior displays both sequential and cyclic transitions between modules offers an extension to our understanding of the flexibility of parallel models in producing different types of serial behavior. We found that fruit flies make frequent transitions between body cleaning and leg rubbing and that these oscillations can be explained by the same hierarchical rules as those that govern the sequential progression of the behavior . The onlDoes fruit fly grooming behavior provide insight into the organization of more complex motor sequences? Our model of the grooming progression has features like those proposed to explain learned sequences that also require parallel activation of units that compete for motor output. An example of this was derived from studies of human typing that revealed that adjacent letters in a word were often exchanged in error. This suggested that a plan for all the letters necessary to generate a word was readied prior to typing and that execution of each letter competed for output based on a hierarchical prioritization scheme and winner-take-all competition . Such stIn this study, we took advantage of the modularity of grooming to interrogate its behavioral organization. We gained the experimental control necessary for probing this organization by identifying GAL4 lines that target neurons whose activation drove specific cleaning modules. We show cause and effect experiments indicating that suppressive interactions among the modules govern their selection. Many behaviors are thought to consist of subdivisions of simpler motor programs. This approach of acutely manipulating subcomponents of behaviors and assessing how they affect the other components may prove useful for exploring the organization of other complex motor behaviors.Our results do not address the neural circuits that mediate the grooming progression but rather suggest possible types of circuit organization that could underlie its sequential nature. Many complex behaviors are likely to involve hundreds or even thousands of neurons and information about the behavioral organization helps focus circuit-mapping efforts on specific features. For example, one prediction of our model of hierarchical suppression points to unidirectional lateral inhibitory connections among the cleaning modules . PreviouUAS-Shibirets1 or UAS-TNT were unable to block cleaning (unpublished data). This may be explained if the GAL4-targeted neurons are those referred to as ‘command-like’   . These nfruitless gene is expressed in 1500 neurons of different types that comprise elements of a courtship circuit  was obtained from Paul Garrity at Brandeis University (UAS-mCD8::GFP (JFRC7) was constructed as described in Canton S flies were obtained from Martin Heisenberg's lab in Wurzburg, Germany. GAL4 stocks were generated in Gerry Rubin's lab at Janelia Research Campus by methods described previously  and are iversity . 20xUAS-Multiwell grooming chambers: blocks of grooming chambers were designed to facilitate the parallel and uniform dusting of flies for dust pattern measurements and recording videos of cleaning behavior. Blocks were fabricated with 24 or 96 grooming chambers (15.6 mm or 7 mm diameters respectively) and have connector ends and dust removal ends. The connector end has tracks for thin aluminum sliders that gate each chamber to facilitate fly loading : aluminum chambers were identical to those described above, with the exception that the dust removal end was closed such that flies would stand directly on the aluminum (shown in  loading . The dusshown in . These c2. 0-min time samples were obtained by dusting the flies and immediately anesthetizing them. Data were acquired from at least four separate crosses. 25 min was chosen as the assay end because most body parts were clean at that point.All experiments were carried out in a warm room that was set to 30–32°C. Flies were cold anesthetized (1–2°C), transferred to grooming chambers (single fly per chamber), and allowed to recover for at least 15 min before assaying. Flies were tapped from the chambers into multiwell plates containing measured amounts of Reactive Yellow 86 dust  and shaken to uniformly coat each fly . Excess Heads: flies were decapitated using a razor blade, and the heads were affixed face up to a cover slip with double-sided tape . Wings: the left  wings were removed by grasping the base with a pair of forceps and pulling it off with an adjacent pair of forceps. They were affixed dorsal side up to a cover slip with double-sided tape. Ventral abdomen: dissected and imaged shortly after performing grooming assays because the abdomens desiccated quickly. Flies were held on their sides with a scalpel inserted between the head and thorax while all legs and wings were removed. The scalpel was then used to decapitate the fly. Next, the thorax and dorsal abdomen were glued to a cover slip such that the ventral abdomen was facing up using Elmer's No-Wrinkle Rubber Cement . Dorsal abdomen: flies were held on their sides and the ventral side of the thorax and legs were severed from the dorsal side along a plane that was parallel to the abdomen. The wings were removed and the abdomen was glued dorsal side up. Thoraces: no dissection was necessary for imaging thoraces. They were imaged in specially designed chamber that would hold all flies uniformly dorsal side up. Standard preparations and example dirty samples for each body part are displayed in Dusted flies were prepared as described above and dissected for imaging of their dust patterns. The dissection procedure made it impossible to image the entire body surface of a single fly. Each part was dissected as described below. Body parts were imaged using a Zeiss SteREO Discovery.V12 equipped with an Achromat S 1.0× objective . Heads, abdomens, wings, and thoraces were imaged at 80×, 60×, 65×, and 60× respectively. Z-series were taken and the images were converted into a single, in focus, image with Zeiss AxioVision software using the Extended Focus module . ExperimCalculating normalized dust pixels in a specified region: pixels with 255 grayscale values (dust pixels), and located within the regions designated by standard masks were counted for each image  equipped with a Computar 25 mm f1.4 lens and an EX2C extender for C-mount  was used to record flies at 30 frames per second. dTrpA1 behavioral analysis (undusted flies): this method was used for recording the flies shown in Dusted experiments: Canton S or GAL4/UAS-dTrpA1 flies were dusted individually in 24-well grooming chambers as described above. An acrylic plug was inserted into the hole on the adapter end and the aluminum slider was removed such that the fly could be recorded . A list of cleaning movements was compiled and modified from two different studies . Flies cEye and head cleaning: legs sweep across the eyes, tops, and bottoms of the head as it is rotated. Most sweeps across the eyes occur in the absence of head rotation. Antennae: legs are directed towards the antennal region, often while the head is tilted slightly forward. The legs sometimes grab the antennae and pull them away from the head. Proboscis: sweeps down the length of the extended proboscis. The amount of proboscis extension does not factor into the definition as long as it is partially extended or seen moving with the legs. Ventral head: legs sweep the ventral side of the head with no proboscis extension. The front legs almost always sweep the bottom of the head/proboscis in parallel. Note, proboscis and ventral head cleaning were binned with eye and head cleaning for simplicity. Whole head: all of the head cleaning behaviors described above are binned. The whole head designation was used because it was not possible to distinguish between eye, head, and antennal cleaning when flies were dusted.Abdominal cleaning: legs sweep the ventral or dorsal surface of the abdomen. The hind legs sometimes clean the genitals. This was included as abdominal cleaning for simplicity. Wing cleaning: each leg sweeps the ipsilateral dorsal side of the wings simultaneously, or one at a time. Alternatively, both legs clean a single wing with the ipsilateral leg cleaning the dorsal side and the contralateral leg cleaning the ventral side. Sweeping or kicking the ventral part of the wing with one or both legs was also referred to as wing cleaning. In this case, as the legs sweep anterior to posterior, the wings lift away from the body. Thoracic cleaning: hind legs sweep the thorax from anterior to posterior with one or both legs.Front/hind leg rubbing: distal parts of the legs are rubbed together. Behavior begins at the moment that the legs touch each other. If the legs stop motion but remain together, it is defined as leg rubbing as long as they do not touch the ground. The middle legs were sometimes rubbed together with the front or the hind legs. This was included with front leg or hind leg rubbing depending on the leg pair being used with the second legs.Standing: fly does not move more than a body length from the original scored position and does not perform any grooming movements. The beginning of standing is scored as soon as the last leg touches the ground. In some cases, the legs did not touch the ground to initiate the behavior and instead was scored at the point where the previous behavior was judged to have ceased. Walking: fly steps more than a body length with no pauses in leg movement. Walking is also defined as moving from one of the following locations in the chamber to another . The beginning of walking is scored at the moment that one of the front legs initiates a step.Bout frequency: total number of starts of each behavior per fly. Bout frequency per interval: bout starts that fell within a designated time interval. Marginal probability: the average fraction of total bouts of a particular behavior. Change from control: the experimental parameter (either bout frequency or fraction of time) was subtracted from the control. Statistical test: control and experimental flies were compared using a Kruskal–Wallis test. Post-hoc Mann–Whitney U pairwise tests were then performed with Bonferroni correction. Transition probability: calculated by counting the number of transitions from each behavior to each other behavior. Next, we calculated the fraction of times that behavior i transitioned to behavior j given the total of number of transitions to all behaviors. Therefore, the probability of going from behavior i to all other behaviors sums to 1.Manually scored behavioral video was analyzed as follows. UAS-dTrpA1 (UAS-Shibirets1 or UAS-TNT (data not shown). GAL4 lines expressing GFP were stained and their patterns imaged using previously published methods . For the UIM architecture, the activation levels are initially set to equal the vector of amounts of dust, d (this is the sensory input layer). The initial amount of dust was set to 0.9 + r, where r is a random value between 0 and 0.1, sampled from uniform distribution. This noise term is only a factor in s, where ws is the vector of sensory weights. In UIM, ws is set to one (no sensory gain differences) and vector a is then multiplied by the N × N weight matrix, W, of which the diagonal = 1, and the upper triangle is populated by equal negative weights, w = −0.5. For SGM, ws is set to ws = , which insures that the sensory weights decrease from the first module to the last. Thus, for each iteration i, we update the activation levels as follows:s to 1, and the upper triangle of W to 0.For simulating no hierarchical differences among the modules shown , we set Winner-take-all layer: to simulate removal of dust from the body parts, this layer first finds the module with the highest activation level that was set by SGM or UIM. The position of the module with maximal activation level, ma, is determined: ma = max(a). The winner-take-all layer is then simply a binary vector e containing all zeros and equals 1 only in the position ma, thus pointing to the body part where cleaning behavior will be executed. Next, a constant amount of dust, dr, is subtracted from the math position in vector d (dma). Thus, for each iteration i:ma) by simply using the Matlab max function. Winner-take-all neural networks can be modeled using all-to-all inhibitory connections between the different modules  (t level) . This moLeg rubbing: to implement leg rubbing, we assumed two pairs of legs. Both pairs were assigned an initial activation level , which was directly proportional to the amount of dust on legs (dl). For each iteration i, we added a small, constant amount of dust to the pair of legs assigned to clean the body part with the maximal activation level (ma). If ma =< 3 (‘anterior body parts’) the first leg pair was assigned and if ma > 3 (‘posterior body parts’) the second leg pair was assigned to collect dust. Therefore, with each grooming iteration, the activation level of the associated pair of legs increases.ma), including the other pair of legs. The latter condition assures that leg rubbing is still part of the competitive hierarchy of cleaning modules  is initiated under two conditions: (1) the activation level of the pair crosses a stationary threshold (same for both pairs) and (2) the activation level of the pair is above the activation level of any body part (above  modules .Model output and display: the output of the model is the position of the cleaning module with the maximum activation level at iteration i(mai). Thus the output is an array with rows corresponding to mai (active cleaning module) and columns corresponding to iteration i (‘time’). This array was used to generate the ethograms shown in  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  VijayRaghavan (Senior editor) and 3 reviewers, one of whom, Ronald L Calabrese, is a member of our Board of Reviewing Editors.Thank you for sending your work entitled “A suppression hierarchy among competing motor programs drives sequential grooming in The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.The authors describe experiments on fruit fly grooming when the entire body is dusted. This paradigm sets up a distinctive motor sequence in which grooming proceeds and allowed the testing of the organizational principles governing how dusted flies groom their bodies with sequential movements. Using genetically targeted activation of neural subsets, the authors drove different components of the grooming sequence that clean individual body parts. They then performed competition experiments to show that the different motor components are organized into a suppression hierarchy so that components that occur first suppress those that occur later. They then make a simple 'neural' model with two layers, a hierarchical layer of the independent cleaning movements and a winner take all layer, which nicely captures their data. Cleaning one body part reduces the sensory drive to its cleaning movement, which relieves suppression of the next movement, allowing the grooming sequence to progress down the hierarchy. They then discuss the implications of their finding for how sequential behaviors are performed and how simple behavioral choices are made.The paper is beautifully illustrated and all necessary data are presented in the figures and figure supplements. The writing is concise and in general clear, and the Discussion is illuminating, but could be expanded a bit. The paper is highly interesting and innovative and provides a mechanistic explanation for how motor sequences can be organized. It should be of wide interest to the neuroscience community.Both the outside reviewers and the Reviewing editor found this paper a pleasure to review and the authors are to be commended for an exceptional piece of work beautifully presented. We encourage the authors to integrate some of the supplemental data into the text, particularly The outside reviewers had several concerns that are intended to make this interesting paper yet more interesting. The reviews are very complementary and require only some rewriting to add details, some more discussions of implications and limitations, and limited amount of further analysis. We anticipate that the authors should be able to quickly answer these concerns, despite their considerable number and resubmit this exciting work. No new experiments are requested.Specific comments:Reviewer #1:The paper is very cleanly written.I encourage the authors to integrate some of the supplemental data into the text, particularly Methods are a little terse; particularly the models and their outputs could be explained more fully.Reviewer #2:This is an altogether impressive piece of work. The approach, although incredibly complex in its details, is extremely clear-headed in how it is organized and presented. The results are spectacular and, at least to me, completely unexpected. They screened more than 1500 Drosophila GAL4 stocks created at Janelia that expressed a temperature-sensitive cation channel (dTrpA1) in all the GAL4-expressing neurons. From these, they found 12 lines that reliably affected different aspects of grooming behavior. They then devised behavioral and anatomical tests to assay the sequence of behaviors that normally occurs in normal grooming. They then used activation of the components of grooming (using the legs to wipe dust from different body parts) to test whether the sequencing of the grooming components  depends upon a series or a parallel mechanism.  Both the behavioral and genetic tools they devised are extremely clever , and the analyses are appropriate and convincing. The major conclusion  is well documented and, for the most part, clearly presented.There are, despite these great strengths, a few problems with the study. Some of these problems are mentioned briefly, but others are not. The major ones are:1) We are told nothing about the behavioral phenotypes of the 12 lines at the elevated temperature. Ideally, it would be nice to know that the obsessive activation of some aspect of grooming is the only behavioral anomaly that they show.2) From the extreme variety of neurons that express GAL4 in the different Drosophila lines , it is remarkable that these lines express such specific behavioral activations when heated. I was disappointed that there was no discussion of this remarkable observation. I could think of several alternatives, but was disappointed that people who are much more familiarity with these animals did not address this question, at least briefly.3) As far as they carried it, the modeling study was quite useful. Having a fairly simple model that matches the data so well, even some data that they didn't use to construct the model is very comforting. They made a big point, however, that there were two possible explanations  for their results, but they attempted to model only the parallel one. Showing that any serial model has serious problems would have rounded off the argument in a satisfying way. They discuss why the serial option would probably not work, but I found this Discussion unconvincing.4) I felt blind-sided when, in the Results, I was told that “the frequent alternations between cleaning the body parts and leg rubbing … are an important and prominent feature in normal grooming behavior”. If these movements are so important, why are they mentioned only as a second-order test of the model and not in any of the studies of the behaviors? Minimally, at the end of the first paragraph in Results the authors could tell us that there is another feature of grooming that will be detailed later. In fact, the initial transitions diagram  has a hi5) They mention that silencing the same GAL4-expressing neurons does not eliminate the behaviors that activating them elicits. Again, I was looking forward to their ideas about why these neurons are sufficient but not necessary, but they did not go there.Reviewer #3:eLife with some modest changes I describe below.Understanding how specific sequences of behavior are executed remains challenging, with a number of alternative models fitting data in different contexts. In this work, Seeds et al. describe a new behavioral sequence that underlies grooming behavior, and take advantage of quantitative analyses and genetic manipulations that allow them to get at the organization of this behavior. Intriguingly, these studies support a parallel track model in which sensory input, combined with different sensory gains and a winner take all competition recapitulate many of the core features of fly behavior. As this system is the first to capture a sequence in which the sensory input that drives the behavior is the same across modules, it represents a powerful paradigm for the dissection of its neural control. This work is quantitative, rigorous, makes a general point and is appropriate for My only concern regarding the fly behavioral data is that I believe that My second concern has to do with the explanation of the structure of the models the authors use. As far as I can tell, the model structure using sensory feedback and the winner take all layer should be deterministic – at whatever level of sensory gain is initiated, combined with the present level of dust, only one module should be selected (and should persist until the level of dust on that body part drops sufficiently for another module to win). However, the data in  Reviewer #1:The paper is very cleanly written.I encourage the authors to integrate some of the supplemental data into the text, particularlyandeLife’s online format should facilitate this.We intended for the main figures to provide a concise view of the results, while the supplemental material expands on the complexity of the data. We made the following additions to the text and figure legends to insure the reader receives full benefit of the figure supplements. Particularly we have tried to make it easier to logically link to figure supplements from the Results section and the primary figure legends. The ease of viewing those figure supplements that complement the primary figures in “The legend describing “Data shown here is compiled and plotted in The Results describing Methods are a little terse; particularly the models and their outputs could be explained more fully.We have changed and added text in the methods sections to improve clarity. Sections modified include: Grooming apparatus; Dusting flies for body surface imaging or video recordings; Quantification of body surface dust patterns; Behavioral recordings; Movement scoring guidelines and definitions; Computational model of hierarchical suppression among competing cleaning movements. The Methods pertaining to the model were expanded to describe the outputs and to provide greater conceptual linkage to the Results presented in Reviewer #2:1) We are told nothing about the behavioral phenotypes of the 12 lines at the elevated temperature. Ideally, it would be nice to know that the obsessive activation of some aspect of grooming is the only behavioral anomaly that they show.We deliberately chose lines for this study that were extremely specific for grooming behaviors. These lines were assessed for gross behavioral phenotypes and motor coordination. This was done both in the course of their selection while screening the GAL4 collection and in doing the experiments in this paper. These lines constitutively clean particular body parts and show no other obvious behavior. One line shows a slight motor impairment when it is walking . We emphasize that the readers can watch the representative videos of different GAL4/dTrpA1 lines to see what the phenotypes look like. We added two additional videos  so all of the lines used in the experiments in We also reference these videos in the Results section of 2) From the extreme variety of neurons that express GAL4 in the different Drosophila lines , it is remarkable that these lines express such specific behavioral activations when heated. I was disappointed that there was no discussion of this remarkable observation. I could think of several alternatives, but was disappointed that people who are much more familiarity with these animals did not address this question, at least briefly.Drosophila fruitless circuit). We also described what the actual cell types might be in this discussion section. Namely we discuss two possible neural types as capable of activating such specific behavior: 1) sensory neurons 2) Command-like/decision neurons.We now address the expression patterns targeted by the GAL4 lines in the last Discussion section that also covers comment 5 from this reviewer (last three paragraphs of the discussion). In brief, we make two points about these expression patterns as they relate to the behavioral activation phenotypes: 1) Only a fraction of the cells in each expression pattern are responsible for the phenotypes as GAL4 lines do not necessarily target functionally related neurons. 2) The cells targeted in each pattern may be related in function (e.g. such as in the 3) As far as they carried it, the modeling study was quite useful. Having a fairly simple model that matches the data so well, even some data that they didn't use to construct the model is very comforting. They made a big point, however, that there were two possible explanations  for their results, but they attempted to model only the parallel one. Showing that any serial model has serious problems would have rounded off the argument in a satisfying way. They discuss why the serial option would probably not work, but I found this Discussion unconvincing.Two major aspects of our data led us to reject the serial model and focus on computationally simulating the parallel model. First, the cleaning modules have parallel sensory inputs and stimulation of one input does not result in a complete sequence of movements, as might be predicted by an activation chain. Second, a serial model does not predict the return cleaning among the modules that occurs in wild type flies (return cleaning is described more in the next paragraph). We reasoned that an activation chain model would simply result in a deterministic sequence of simulated grooming movements, rather than this return cleaning. This said we make no claim that some degree of internal triggering of cleaning modules (through an activation chain) does not exist. In other words, a ‘mixed model’ could explain the empirically observed grooming behavior even better. In this case, execution of a cleaning module is selected by two components: winner-take-all  and internal triggering from the previous cleaning module . All we claim in this work is that the parallel model alone is fully sufficient to reproduce the major aspects of grooming sequence discussed in this manuscript. We revised the Results and Discussion to better set up and then state our argument against an activation chain as the primary mediator of the grooming sequence. The manuscript has been changed to address this and other related comments from reviewers two and three. We now focus more attention on the return cleaning that occurs among the cleaning modules. We do this by first establishing that the flies clean a body part, then clean the next, but still return to clean previous body parts. These returns by the cleaning modules in wild type flies are now emphasized more in the Results that describe 4) I felt blind-sided when, in the Results, I was told that “the frequent alternations between cleaning the body parts and leg rubbing … are an important and prominent feature in normal grooming behavior”. If these movements are so important, why are they mentioned only as a second-order test of the model and not in any of the studies of the behaviors? Minimally, at the end of the first paragraph in Results the authors could tell us that there is another feature of grooming that will be detailed later. In fact, the initial transitions diagram  has a hint of this alternation; it could be mentioned in discussing this diagram.We apologize that this was not clear and now emphasize leg rubbing more in the first paragraph of the Results section. There was already text in this paragraph that referred to leg rubbing in grooming behavior. In fact, our primary purpose for including To make this point even more clear, we added additional text that mentions these cyclic transitions in the second to last sentence of the first Results paragraph: “Thus, flies groom by gradually cleaning different body parts in bouts that are characterized by cyclic transitions between body cleaning sweeps and leg rubbing (shown as a series of transition diagrams in 5) They mention that silencing the same GAL4-expressing neurons does not eliminate the behaviors that activating them elicits. Again, I was looking forward to their ideas about why these neurons are sufficient but not necessary, but they did not go there.We now address this point in a new (last) section of the Discussion that also covers point two from this reviewer: “Neurons that activate cleaning modules”. In brief, we make the point that the neurons may be sensory or command-like neurons. We describe that such command-like neurons do not necessarily follow a strict definition as command neurons in being both necessary and sufficient. Alternatively (not described in the discussion), redundancy exists among the grooming sufficient neurons, and each GAL4 line only targets a fraction of the total population sufficient to drive a specific cleaning movement. This could obscure results of an experiment to test neural necessity in grooming because the entire population would not be inhibited through expression of neural inhibitors.Reviewer #3:My only concern regarding the fly behavioral data is that I believe thatis oversimplified, and hence misleading. The time traces inand the fraction changing indemonstrate that the transition from one behavior mode to another is quite smoothly graded, a feature that is eliminated from the very simple view in. I would therefore request that the authors extend the type of display in, developing a time-dependent transition probability model. A simple way to do this would be to cut the ethogram data into 5 minute segments, and replot the transition probability model . This kind of representation should give a more accurate sense of how the behavior changes progressively.We agree with reviewer three that Reviewer 3 also suggested that we produce time-binned diagrams like  from theMy second concern has to do with the explanation of the structure of the models the authors use. As far as I can tell, the model structure using sensory feedback and the winner take all layer should be deterministic – at whatever level of sensory gain is initiated, combined with the present level of dust, only one module should be selected (and should persist until the level of dust on that body part drops sufficiently for another module to win). However, the data inshow that, like real flies, at most time points, the models have mixed character, with flies alternating between different activities (which change progressively). I understand how leg grooming is exceptional, in the sense that its sensory drive increases periodically (giving an oscillatory character to the behavior), but for all other grooming behaviors, the sensory drive can only decline. Is there a noise term that is not described in the text? If so, what is its form? If not, can the authors provide a more accessible description for why their models are not deterministic in time?We do use a noise term in our model that varies the initial amount of dust on the cleaning modules in the upper ten percent of the total dust amount (see revised Methods). This causes variability in which modules are selected first when there is no suppression hierarchy (i.e. as in


The UK Biobank offers cross-sectional epidemiological data collected on >500,000 individuals in the UK between 40 and 70 years of age. Using the UK Biobank data, the aim of this study was to investigate the effects of functional hearing loss and hearing aid usage on visuospatial memory function. This selection of variables resulted in a sub-sample of 138,098 participants after discarding extreme values. A digit triplets functional hearing test was used to divide the participants into three groups: poor, insufficient and normal hearers. We found negative relationships between functional hearing loss and both visuospatial working memory  and visuospatial, episodic long-term memory , with the strongest association for episodic long-term memory. The use of hearing aids showed a small positive effect for working memory performance for the poor hearers, but did not have any influence on episodic long-term memory. Age also showed strong main effects for both memory tasks and interacted with gender and education for the long-term memory task. Broader theoretical implications based on a memory systems approach will be discussed and compared to theoretical alternatives. There is sufficient evidence to conclude that there is a connection between sensory decline and cognitive decline. Decline in one function is associated with decline in the other and the strength of the association has been empirically shown to increase with increasing age  between different hearing-related perceptual aspects on the one hand and cognitive aspects on the other. One such mechanism is perceptual stress or perceptual degradation, where it is typically assumed that even when stimuli are audible, the hearing loss affects the quality of encoding of memory items , that the locus of the effect is at the level of memory systems, and that there is selectivity in terms of which system is most affected. We outline the reasons for the predictions below:combined the degree of hearing loss with the degree of visual acuity did not make satisfactory predictions of memory decline for any memory system. Thus, the results suggest that relative decline in a memory system is tightly connected specifically to hearing loss rather than to sensory decline in general.In Rönnberg et al.  it was fRönnberg et al.  explaineThe prediction regarding semantic long-term memory based on a use/disuse concept is less clear because it could be argued that semantic and contextual knowledge would have to be used more than episodic memory to compensate for misheard or non-matching words  of people not using hearing aids from the much larger UK Biobank Resource , we therefore focused on the effects of hearing loss and age on memory tasks that were not confounded by possible auditory perceptual degradation, or by attentional demands related to hearing difficulties, strictly testing the memory systems hypothesis.In the current study, based on a large sample  after having had a short inspection time. Two versions of the task were employed, an easy one with three pairs (which was considered to be a warm-up task), and a more difficult one with six pairs . Thus, we opted for the six pair version in our analysis to maximise the demands on working memory.blue square, but as a prospective memory test, they should instead touch the orange circle.As a proxy for episodic long-term memory function and to determine whether we could replicate the negative effect of hearing loss on episodic long-term memory , we also checked for the effects of hearing aid usage, with the hypothesis that this may have a protective effect against memory decline . This resulted in a study sample of 138,098 participants. Among these, 75,065 were females and 63,033 were male; giving a slightly skewed ratio of 54/46 (%). Age ranged from 39–70 years reflecting the UK Biobank population as a whole.N = 439,510) and “difficulty following a conversation if there is background noise ” . Among the UK population, 114,717 (25%) reported having general difficulty with hearing, 169,055 (37%) had difficulty hearing in noise, and 14,010 (3%) wore hearing aids. In our sample of 138,098 persons, we had data for 130,206 on reported general difficulty with hearing, and 24% reported such difficulty. For hearing in noise, we had data for 134,673 persons and 34% reported difficulties with that. With respect to hearing aid usage, 3751 persons (2.6%) in our sample reported wearing a hearing aid.The UK Biobank population also answered yes/no questions about “difficulty with hearing in general”  and whether they had diagnosed eye problems/disorders other than those corrected for by the use of glasses. In our sub-sample of 138,098, 89%  reported having eye-glasses and 88%  reported having no additional eye-problems.Participants were also asked which of six qualifications they had obtained. To simplify further analyses, a new highest level of qualification variable was created that assumes that a College or University degree (rated 1) > A levels/AS levels (rated 2) > O levels/GSEs (rated 3) > CSEs (rated 4) > NVQ or HND or HNC (rated 5) > Other professional qualifications; e.g., nursing or teaching (rated 6). In our sub-sample, we had valid values for 116,947 on qualification and the distribution across qualification levels 1–6 was 38.8%, 13.8%, 26.6%, 7.1%, 7.7%, and 6.0%, respectively.The study presented here is covered by a Research Tissue Bank approval obtained by UK Biobank from its governing Research Ethics Committee, as recommended by the National Research Ethics Service.Participants attended 1 of 22 assessment centers spread throughout the UK. All test data used in this study were obtained through a self-administered program running of a computer with a touch screen that collected responses to questionnaires and tests on hearing in noise and cognition. Incomplete data sets were collected as it was possible for participants to be selective in which questionnaires and tests they responded to.The participants completed a functional hearing test in which they were presented with digit triplets in a steady state, speech-shaped noise  and hence perform differently when listening to speech in noise. Therefore, it seems reasonable that DTT also has been found to correlate highly with speech-in noise-recognition measures  Prospective Memory test: Shape—Part 1; (2) Pairs memory test; (3) Verbal Reasoning test; (4) Reaction time: Snap; (5) Prospective Memory test: Shape—Part 2. We here describe the pairs matching and the prospective memory tests, as they are used for the short-term—long-term memory distinction relevant to this paper. Data on reverse digit span were also available from the UK Biobank resource but were not used in the present study with its focus on visuo-spatial memory function.VSWM was measured with a pairs matching game. Participants were presented first with a round of three pairs of cards depicting different designs of objects and then, twice, with a round of six pairs of cards. The layout was purely random each time. There were no specific selection criteria applied to choosing the designs of the pictures other than that they should look reasonably distinct. Thus, there were no systematic phonological or semantic relationships between the English lexical labels of the pairs of objects. During each round, the pictures were turned over after a short inspection period. The 2 × 3 layout was shown for 3 s before pointing and the 2 × 6 layout was shown for 5 s. The participants were asked to identify as many pairs as possible with the fewest attempts by touching “pairs” of the same object on the screen. When the participant made an error, this was indicated by the word “miss” appearing at the center of the screen. When the participant gave a correct answer, the word “pair” would appear on the screen. For each correctly identified pair, the cards were removed and two blank spaces were left in the position where they had previously been placed. The participants could continue until they had identified all pairs. Time allowed for matching of pairs was unrestricted. The participants were allowed to continue until they had discovered all pairs correctly. The dependent variable is thus the number of errors made before all the pairs had been matched. We considered the three-pairs round a warm-up trial for the six pairs round, which constituted the dependent variable.PLTM consisted of two parts:Part 1. The initial instruction to the participant was the following: “At the end of the games we will show you four colored shapes and ask you to touch the Blue Square. However, to test your memory, we want you to actually touch the Orange Circle instead. Once the “Next” button was touched, a hidden timer was started to record the delay interval until the answer to this question (asked after the reaction time test) was requested. Then the Pairs matching test, the Fluid intelligence test and the Reaction time (Snap) test were performed.Part 2. After the Reaction time (Snap) test was finished, the following text was shown to the participant: “That’s the last game. Just one more thing left to do…”. The participant then selected “Next”; and the Shapes screen appeared with the text: “Please touch the Blue Square then touch the “Next” button” was presented. At this point the delay interval timing ended. If the participant touched any of the symbols it was highlighted by surrounding it in a yellow box. If the participant touched the Next button without having highlighted a symbol they were shown the message: “Please touch a symbol (a colored shape) before touching the “Next” button” If the participant then touched any symbol other than the Blue Square, then Next, the test ended. If the participant touched the Blue Square, they were prompted with the message: “At the start of the games we asked you to remember to touch a different symbol when this screen appeared. Please try to remember which symbol it was and touch it now”. If the participant touched the Blue Square again then this message was repeated (ad infinitum), otherwise the program accepted their new selection and the test ended. The dependent variable was scored in three steps: correct at first attempt, correct at a subsequent attempt, and not correct at first or following attempts .x + 1) to counteract the skewed distribution of the raw scores. Also, for the analyses of the VSWM and PLTM tasks, individuals with values above the 99th percentile on the six pairs matching tasks were excluded to build in a safeguard against outliers. Our initial analyses were also restricted to participants who did not use hearing aids.For the memory measures, logarithmic transformation of the number of errors made in VSWM and the errors scores in PLTM were computed , as suggested by Dawes et al. ; see alsN in the smallest Age × Hearing status groups were ≈ 100 or more. With these criteria our oldest group was defined as >67 years, and the youngest as <48 years, with two 10-year age spans in between.We preferred four age spans, and that the two middle spans would be 10 years. With hearing status groups already defined, the pragmatic solution was to move the two middle 10 year age spans down from the maximum age of 70 years in our sample, and ensure that the N = 138,098 in our subsample. Table The Age by Hearing status distribution is shown in Table Table We also decided to take a parametric approach to how to treat the logarithmic error scores for VSWM and PLTM. The basic issue is whether it can be justified to treat the scores as being on an interval scale, and reanalyze them with parametric tests, such as ANOVA, or whether the data only meet ordinal scale properties and should thus be subjected to non-parametric tests. We concluded that an ANOVA approach is justified, but we will discuss the pros and cons of that at the end of the Results Section and also provide non-parametric analyses of our data to support the parametric statistical analyses.F-values for the two memory tests, but the effect of Hear for PLTM appears to be stronger than it is for VSWM. Also, there is a significant interaction Hear × Age for VSWM, but not for PLTM.Figure F = 68.9, p < 0.000, between the normal and insufficient hearers, F = 256.6, p < 0.000, and a significant effect of Age, F = 12.18, p < 0.000, but no significant effect of their interaction (F < 1). For VSWM, there was no significant difference between the poor and insufficient hearers, (F < 1), a main effect of Age, F = 12.81, p < 0.000, and no significant interaction (F < 1).Thus, PLTM seems to be more sensitive to functional hearing status and judging from Figure Thus, the ANOVAs and the pattern of simple main effects results strongly support the conclusion that there is a crucial difference in the pattern of age-related performance between PLTM and VSWM, especially when comparing the poor and insufficient hearers. Poor compared to insufficient hearing is markedly more deleterious to PLTM than it is to VSWM.In Table As shown in Table Therefore, effects sizes are quite in line with the results from the separate ANOVAs, which showed large effects of both Hear and Age, the Age effect being about equal for the VSWM and PLTM, but also that the Hear effects were larger for PLTM than for VSWM.To assess whether using a hearing aid modulated memory decline, we computed separate ANOVAs on the following sub-sample: for a total of 3751 of HAUse we had data on their Age and Hearing status, as well as on their scores within the 99th percentile on the memory tasks. Of these, 2139 were normal hearers , 1080 insufficient hearers (29%), and 532 were poor hearers (14%).F = 4.86, p < 0.05). For VSWM there was also a significant interaction Hear × HAUse, F = 4.20, p < 0.05, see Figure F = 7.10, p < 0.01 (with a Cohen’s d effect size of = 0.185) but not at the other two levels of hearing (F < 1). Thus, for VSWM the results indicated that for the normal hearers there was not much of a difference between those with and without hearing aids, but with increasing hearing loss the degree of “protection” against memory errors afforded by wearing hearing aids increased , and no significant interaction Hear × HAUse (F < 1), but there was a significant interaction Age × HAUse, F = 6.05, p < 0.000, which was specified by the interaction Hear × Age × HAUse, F = 3.06, p < 0.01, (not given in any figure) showing that the poor hearers with hearing aids in the youngest group have markedly higher error scores than was the case for the other HAUse . Thus, generally speaking, PLTM was not positively affected by the use of hearing aids, but for VSWM we could observe some more “protection” against making errors, as is suggested from the HAUse × Hear interaction in Figure For PLTM, there was no main effect of HAUse,  = 85.19, p < 0.001, and an interaction Hear × Education, F = 7.73, p < 0.001. These effects indicated that the persons with a lower education made more errors, and that this disadvantage was more marked for those with poor hearing. The 95% confidence interval for the poor group included the insufficient group for those with a higher education, but for those with a lower education, the insufficient group was lower by far in errors and outside the 95% confidence interval for the poor group. However, we cannot be conclusive about education causing better episodic long-term memory, but there are studies that suggest that schooling affects brain function and cognition many decades after schooling has terminated  = 2.74, p < 0.05, meaning that males with lower education and in the age range 48–57 years, made more errors than women in the same group. However, caution should be observed when interpreting these results as the number of persons in 4 of the 48 (= 4 × 3 × 2 × 2) cells come as low as n < 30, particularly for the youngest and oldest poor hearers with high education.For PLTM, there was also an interaction Age × Gender × Education, Fs < 1.97).Furthermore, replacing Hearing status in the original ANOVAs with the binary scored subjective reports of hearing difficulty and hearing difficulty in noise, did not yield any significant main effects or interaction  that accounts for the observed memory declines.To safeguard against missing some more delicate and detailed effects when a rather crude hearing criterion like the three-step Hear-distinction was employed, an analysis with a four-step hearing criterion (Hear4) was also performed. In this four-step criterion the extreme groups were the same as in the original Hear4 criterion, but the former middle-group (Insuff) was split into two groups, Insuff1 (SRT −5.5 to −5.0) and Insuff2 (SRT > −5.0 to 3.5). The number of persons are shown in Table The results of the Hear4 grouping is depicted in Figure As can be seen when comparing Figure N = 1 340) was divided into three poor hearing groups . The results are shown in Figure F = 3.25, p < 0.05. For PLTM there was no significant effect of Age (p > 0.10), but as indicated in Figure t-test, t(789) = 1.78, p < 0.05, but Cohen’s d was low (0.127).In a similar vein, we also probed what would happen to the scores for VSWM and PLTM when the group with poor hearers N = 1 30 was divThus, a more fine-grained sub-grouping of the poor hearers pinpoints the extremely poor hearers, the worst group, as responsible for a significant share of the increase in error scores for PLTM, but not to the same extent for VSWM.The general result of these analyses is that the effects of functional hearing loss are robust and prominent for episodic long-term memory, and driven by extremely poor hearers. Being a hearing aid user had no effect on the association between hearing and episodic long-term memory, but did influence the association between hearing and working memory; hearing aid users among poor hearers performed better than non-users. Education and gender modulated the episodic long-term memory decline but not working memory. Age affected both memory systems negatively, but interacted with gender and education only for episodic long-term memory.There may be some doubt as to whether the scale properties of our measures of VSWM and PLTM meet the assumptions for a parametric ANOVA-test.However, ANOVAs are known to be robust against violations of the underlying assumptions , while the scale for VSWM was number of errors on and interval scale from 0 to 15. Thus, there was a substantial underestimation of the actual number of errors made in the PLTM task. In spite of this underestimation, poor functional hearing turned out to be significantly related to PLTM, which makes the result even more striking in light of the main hypothesis of the present paper.The focal finding of this study is that functional hearing loss is clearly related to visuospatial episodic long-term memory (PLTM). This result is important for several reasons.First, it shows that the negative effect of functional hearing loss is not restricted to mechanisms coupled to auditory perceptual degradation  effect on the visuospatial working memory task but not on the episodic long-term memory task, the results mimic the Rönnberg et al.  data in A counterargument against the positive effect being due to the use of hearing aids as such would be to reverse causality as follows: if good memory were causing people to get and use hearing aids, the group with normal functional hearing who used hearing aids would have better memory. However, since this was not the case  rests on the fact that functional hearing loss affects PLTM and hearing aid benefit VSWM, i.e., both variables affect the two memory systems selectively. In this study, it happened with a visuospatial VSWM task, but similar results could have been found with an auditory WM task, that is, the general picture that is emerging is that of multimodal processing. The important aspect is the difference in basic cognitive mechanisms underpinning the two tasks, and how other variables latch on to the different properties of those two memory systems.restricted to VSWM and the effect size was also low. This is also in general agreement with Rönnberg et al. , and that this decline is comparable to loss of brain volume in participants with diagnosed mild cognitive impairment  model in the relationship between hearing loss and working memory on the one hand, and episodic long-term memory on the other for different types of tasks . If the short-term or working memory component was crucial, one would then predict that DTT performance should co-vary with VSWM and not with PLTM. Digit triplet test performance did not co-vary with VSWM. The reason for the lack of an association with VSWM could be that a “load” of a digit triplet is clearly below what is typically given as the normal digit span size . Instead, the DTT variable predicted a decline in PLTM. This kind of double dissociation represents evidence in favor of an interpretation of the present results in terms of a negative effect of functional hearing loss on episodic long-term memory, as outlined by the ELU model  acts as a reminder of the set of available items. Fourth, it is currently unknown how central and peripheral auditory factors play out in the DTT. Further research is needed . Also, the range of the raw values of number of errors were three for PLTM  and 16 for VSWM (0–15). The logarithmic ranges and means were: VSWM range 0.00–2.77, mean 1.40–0 errors = 7.1%; PLTM range 0.69–1.39, mean −0.78–0 errors = 80.6%. Again, the PLTM task was less difficult than the VSWM task, had fewer steps, and was less sensitive, but still produced significant differences with substantial effect sizes due to functional hearing loss. Reliability estimates are not available from the UK Biobank resource. If we had observed the opposite pattern, viz. that functional hearing loss was associated with larger effects for the VSWM task, then it could have been argued that the effect  was due to greater task difficulty that provoked the negative memory effect. In all, it seems unlikely that aspects related to task difficulty could explain the results obtained in the current study.Another concern that may be raised against the selectivity in the effect of hearing loss on memory systems is the possibility that the results may be confounded by not related to subjective ratings of hearing disability collected in the UK Biobank database, which suggests that the obtained effects may be based on the loss and objectively determined by an audiogram or by an objective test such as the DTT  than for working memory or short-term memory . This is in line with the ELU prediction about mismatch and relative use/disuse of memory systems (Rönnberg et al., 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.


TCs have very long prolongations (tens to hundreds micrometers) named Telopodes (Tps). These Tps have a special conformation with dilated portions named podoms  and very thin segments (below resolving power of light microscopy), called podomers. To show the real 3D architecture of TC network, we used the most advanced available electron microscope technology: focused ion beam scanning electron microscopy (FIB-SEM) tomography. Generally, 3D reconstruction of dermal TCs by FIB-SEM tomography revealed the existence of Tps with various conformations: (i) long, flattened irregular veils (ribbon-like segments) with knobs, corresponding to podoms, and (ii) tubular structures (podomers) with uneven calibre because of irregular dilations (knobs) – the podoms. FIB-SEM tomography also showed numerous extracellular vesicles  released by a human dermal TC. Our data might be useful for understanding the role(s) of TCs in intercellular signalling and communication, as well as for comprehension of pathologies like scleroderma, multiple sclerosis, psoriasis, etc.We have shown in 2012 the existence of telocytes (TCs) in human dermis. TCs were described by transmission electron microscopy (TEM) as interstitial cells located in non-epithelial spaces (stroma) of many organs (see  Their presence in human dermis was confirmed i) the aspect of TCs and fibroblasts is not the same in tissue culture ii) transmission electron microscopy (TEM) shows completely different ultrastructure, e.g. iii) microRNA imprint is dissimilar, e.g. iv) gene profile are not the same v) proteomics showed striking differences Telocytes (TCs) were described 5 years ago Focused ion beam scanning electron microscopy (FIB-SEM) is now the election technique for three-dimensional (3D) visualization of biological structures (cells) at nanoscale resolution Here, we present FIB-SEM tomography of human papillary dermis TCs showing their complex 3D architecture, as well as the budding and shedding of extracellular vesicles. FIB-SEM tomography does not contradict TEM, but provides additional important details.4 with 1.5% K4Fe(CN)6 (potassium ferrocyanide – reduced osmium) to increase the membranes contrast. Subsequently, the samples were dehydrated through increasing graded ethanol series and embedded in epoxy resin  at 60°C for 48 hrs.Biopsies of human skin were obtained from three patients (informed written consent). Normal skin samples were obtained from a re-excision procedure after removing a local melanoma. The second excisions were performed according to the Breslow index , 14 days after primary excision. The samples of normal skin were taken at 1-cm distance from primary suture Focused ion beam milling and SEM imaging were carried out with a ZEISS Auriga Crossbeam system . FIB milling was performed with 600 pA to 20 nA for the given samples. SEM-Imaging current was 220 pA. To achieve the best signal contrast, the mixed Inlens and energy-selective backscattered detector signals were used. FIB milling steps was 10 nm/slice and each 5th slice was imaged. Accordingly, each image represents 50 nm of the stack, at 9k× magnification. Image pixel size was 10.27 nm.Image stack was analyzed and processed using Adobe Photoshop CS5 Extended  for noise detection and removal and luminance level adjustment. Output images were then loaded into Amira 5.0.1  software package. Structures of interest were manually segmented and reconstructed. Stacks of images were also loaded in VirtualDub v1.10.4 (Lee A.) software as sequence of numbered JPEG files and converted to video file. Adobe Photoshop CS5 Extended was also used for vesicles 2D morphometry. Measurements were then statistically analyzed using Microsoft Excel 2013 Analysis ToolPak module.3  showed a vesicle diameter of 438.6 ± 149.1 nm. Considering the international standards e.g. (i) dimensions over 100 nm, (ii) origin by budding and shedding of plasma membrane and (iii) the monovesicular ultrastructure) In fact, a rough estimation of the number of extracellular vesicles (As previously shown, TCs were found in human dermis having a strategic position: around blood vessels, in the perifollicular sheath, outside the glassy membrane and surrounding sebaceous glands, arrector pili muscles and both the secretory and excretory segments of eccrine sweat glands Last but not least, very recent data suggest that TCs through their Tps could be regarded as a primitive nervous system


Reactive oxygen species (ROS) production is involved in several apoptotic and necrotic cell death pathways in auditory tissues. These pathways are the major causes of most types of sensorineural hearing loss, including age-related hearing loss, hereditary hearing loss, ototoxic drug-induced hearing loss, and noise-induced hearing loss. ROS production can be triggered by dysfunctional mitochondrial oxidative phosphorylation and increases or decreases in ROS-related enzymes. Although apoptotic cell death pathways are mostly activated by ROS production, there are other pathways involved in hearing loss that do not depend on ROS production. Further studies of other pathways, such as endoplasmic reticulum stress and necrotic cell death, are required. Reactive oxygen species (ROS), such as hydroxyl radicals, superoxide anions, hydrogen peroxide, and singlet oxygen are mainly generated by the mitochondria in most mammalian cells , 2. ROS,Elevated ROS formation and subsequent apoptosis induction have been implicated in the development of several hearing loss pathologies . FurtherThe cochlea is the auditory end organ of the inner ear. The organ of Corti is a core component of the cochlea and contains two types of sensory hair cells: inner and outer hair cells. When a sound pressure wave travels from the base to the apex of the cochlea, the basilar membrane of the cochlea vibrates . The disThere are other components and supporting cells in the cochlea which maintain the structure and the environment of the cochlea. The stria vascularis and the spiral ligament are located at the lateral wall of the cochlea and generate the endocochlear potential (EP) . EP is tHearing loss is a reduction in sound sensitivity and is roughly divided into two types: acquired hearing loss and inherited hearing loss. The well-known types of acquired hearing loss are ototoxic drug-induced hearing loss (ODIHL), age-related hearing loss (ARHL), and noise-induced hearing loss (NIHL). The pathological features of each type of hearing loss are different. ODIHL is the commonly irreversible hearing loss caused by the intake of ototoxic drugs and the main mechanism is the loss of the hair cell . NIHL isMany chromosomal genes encoding proteins have been associated with hereditary hearing loss, such as myosin , extraceMitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome  are all The mutation rate of mitochondrial DNA differs between tissues. Rates are higher in the spiral ganglion cells and saccular macula than in the hair cells of the organ of Corti, the stria vascularis, and the facial nerve . There iTwo types of ototoxic drug classes are widely known in clinical practice : aminoglAminoglycosides are broad-spectrum antibiotics that require a close monitoring of their potential ototoxicity and nephrotoxicity . The nepROS are now established as the main initiators of aminoglycoside-induced hearing loss . AminoglThe 1555A>G mitochondrial DNA mutation causes hereditary hearing loss with known aminoglycoside hypersusceptibility . The 12SPlatinum-based anticancer drugs are frequently used in the treatment of many types of cancer including squamous cell carcinoma; adenocarcinoma; and undifferentiated carcinoma of the head, neck, lung, and bladder . HoweverCisplatin has both acute and chronic toxic effects on cochlear tissue. The acute effect is a reversible inhibition of transduction currents and voltage-dependent calcium currents in the hair cells  and the ROS formations in the outer hair cells in response to cisplatin represent the binding of cisplatin to the sulfhydryl group of enzymes and the depletion of nicotinamide adenine dinucleotide phosphate (NADPH), copper, or selenium . These pThe increased ROS generation reduces the antioxidant defense mechanisms of the outer hair cells, causing the release of cytochrome c from mitochondria, activating the caspase pathways, and triggering apoptotic cell death . The cytκB) and the formation of nitric oxide (NO) [κB and inductable nitric oxide synthase (iNOS) immunolabeling [Other potential apoptotic pathways in the stria vascularis of the lateral wall or in the spiral ganglion include the activation of nuclear factor kappa B (NF-ide (NO)  and the ide (NO) . Increaside (NO)  and incrlabeling , 66 havelabeling  and the labeling  indicateThe prevalence of ARHL is expected to increase with aging population , 68–70. The accumulation of mitochondrial DNA mutations is hypothesized to cause age-related degenerative diseases such as ARHL . IncreasBecause ROS play an important role in ARHL , the effBak) [Intrinsic and extrinsic pathways are involved in apoptotic cell death in ARHL. The intrinsic pathway is mitochondrial dependent and is triggered by a loss of the mitochondrial membrane potential. The extrinsic pathway is triggered by ligands that bind to cell surface receptors , 83. FurBak) , indicatNoise is also a major cause of hearing loss . It is oTwo main pathways result in cochlear damage following noise exposure: mechanical damage  and biocSeveral pathways that trigger apoptotic cell death in noise-induced hearing loss have been studied in animal models. Studies have revealed common increases in ROS or similar reactive species  but ROS 2+ in the outer hair cells [2+ and calmodulin-controlled calcineurin [Other pathways which do not depend on ROS production have been predicted. The increase of free Cair cells  or the acineurin  may trigcineurin  caused bcineurin  leads tocineurin .ROS production and mitochondrial apoptotic pathways play important roles in many types of hearing loss. Major ROS production pathways include OXPHOS dysfunction, increased pro-ROS enzyme activity, and decreased anti-ROS activity. Hearing loss pathways vary and some remain under investigation. Other pathways, such as ER stress and necrotic cell death, are also involved in hearing loss. Further studies of each type of hearing loss are required including the investigation of ROS, apoptosis, and other types of cell death.


Dear Editor,Hearing loss is one of the major public health problems. The mitochondrial DNA mutations has been found to be associated with both aminoglycoside-induced and nonsyndromic hearing loss . Furthermore, according to their distinct clusters of mtDNA polymorphisms, we found the mtDNAs of 11 pedigrees belonged to 4 common eastern Asian mtDNA haplogroups A, B, D and F.OR = 2.10; 95% CI = 1.38–3.25; P < 0.01). Furthermore, the increased frequency of 1555A>G mutation on haplogroup D background is attributable largely to specific subhaplogroup D5 . In contrast, those families on haplogroup M8 and G background tended to have a reduced frequency of 1555A>G mutation (P < 0.05).Besides the 11 families with hearing loss above, we collected additional 131 pedigrees carrying 1555 A>G mutation among eastern Asian population from previous publications for further analysis. Of the 142 mtDNAs analyzed, 74 were categorized into macro-haplogroup M, and the rest 68 pedigrees’ mtDNAs were assigned to macro-haplogroup N. mtDNA haplogroup distribution analysis showed that 1555A>G mutation was widely dispersed among 11 major Eastern Asian haplogroups D, M7, M8, M10, M11, G, F, B, R, A and N9 . This observation strongly indicated that the 1555A>G mutation arising independently in different mtDNA haplogroup backgrounds among eastern Asian populations. As shown in Table P = 0.006). Furthermore, when the aminoglycosides-exposed hearing loss cases were excluded, these haplogroup B pedigrees still showed significantly higher penetrance of hearing loss symptom .To further understand the role of mtDNA haplogroup on phenotypic expression for 1555A>G mutation, we evaluated the penetrance of hearing loss among 142 pedigrees (Table S3). A wide range of the penetrance of hearing loss were observed among the 142 pedigrees, with the average of 33.8% and 21.8% when aminoglycosides-induced hearing loss was included or excluded, respectively. Then the clinical penetrance of 1555A>G mutation on specific haplogroup were compared with that on all mtDNA backgrounds


To describe a case of hearing rehabilitation with bone anchored hearing aid in apatient with Treacher Collins syndrome.3 years old patient, male, with Treacher Collins syndrome and severe complicationsdue to the syndrome, mostly related to the upper airway and hearing. He hadbilateral atresia of external auditory canals, and malformation of the pinna. Theinitial hearing rehabilitation was with bone vibration arch, but there was pooracceptance due the discomfort caused by skull compression. It was prescribed amodel of bone-anchored hearing aid, in soft band format. The results wereevaluated through behavioral hearing tests and questionnaires Meaningful Use ofSpeech Scale (MUSS) and Infant-Toddler Meaningful Auditory Integration Scale(IT-MAIS).The patient had a higher acceptance of the bone-anchored hearing aid compared tothe traditional bone vibration arch. Audiological tests and the speech andauditory skills assessments also showed better communication and hearing outcomes.The bone-anchored hearing aid is a good option in hearing rehabilitation in thissyndrome. The Treacher Collins syndrome, first described in 1900 by a British optician EdwardTreacher Collins, is an autosomal dominant disorder that affects one in 50,000 livebirths.TCOF1 (5q32 locus) gene,responsible for encoding the nucleolar phosphoprotein Treacle, which isdirectly involved with the development of the first two pharyngeal arches.,de novo mutation.In this syndrome, there is a mutation in the TCOF1 5q3 locus geThe phenotype of these patients is diverse. There are cases in which the patient ismildly affected, and there may be a difficulty in establishing the diagnosis; on theother hand, some patients have early death in the perinatal period, usually caused bythe severe airway impairment.Between 30% and 50% of the affected children have severe bilateral conductive hearingloss, as a result of stenosis or atresia of the external auditory canal or middle earmalformations.As a result of the malformation of the external auditory canal, the use of traditionalhearing aids, can rarely be indicated. The most common alternative for rehabilitation isthe bone vibration arch. This type of prosthesis has some disadvantages, mainly relatedto excess pressure of the arch, poor esthetical appearance and difficulty in maintainingthe arch in place when the patient is a child, as it can be easily removed .7A recent alternative for the auditory rehabilitation of these patients is the BoneAnchored Hearing Aid (BAHA), which consists of an option in cases of conductive or mixedhearing loss and has a low rate of complications associated with good functionalresults.The present study reports the use of BAHA in hearing rehabilitation of a child withTreacher Collins syndrome with bilateral atresia of the external auditory canal. We alsodescribe the methods used in the auditory evaluation and rehabilitation resultmeasurement for patients with this syndrome and at this age group.Case report based on medical file review, approved by the institution's ethics committeeunder number 24692213.7.0000.0103 and with Informed Consent form signed by the patient'sguardian.The patient was born on September 2010, was male, Caucasian, born and living inCuritiba, Paraná. He was diagnosed with the Treacher Collins syndrome at birth. He had adownward-slanting of palpebral fissure or antimongoloid inclination, malar hypoplasia,micrognathia and macrostomia. He also had microtia and external auditory canal atresiabilaterally.The most severe complications associated with the syndrome were in the upper airways andthe hearing impairment. Tracheostomy was performed soon after birth, and gastrostomy at3 months of age. Orthognathic surgeries were performed at 1 year and 3 months, 1 yearand 8 months and 3 years and 10 months. He is currently stable and well adaptedregarding the airways. Additionally, blepharoplasties were performed at 6 months, 8months, 1 year and 8 months and 1 year and 10 months .At the hearing assessment performed on April 2012, at 1 year and 7 months, the objectiveand subjective tests were used, as described below:Brainstem Auditory Evoked Potential: the child had absent airway thresholds in90dBA and present bone thresholds in 35dBA, on both sides;Behavioral note: this test provides clues and information on global developmentand, especially, the expected auditory behavior for the age of the child, andhelps in the diagnosis of hearing loss and other associated disorders.After these initial assessments, on August 2012, at 1 year and 10 months, a hearing aidwith bone vibrator was adapted. At the test, a functional gain of 40dB was recorded; however,During consultation one month after the BAHA system activation, we applied two protocolsto measure the speech and listening skills: Meaningful Use of Speech Scale (MUSS) andInfant-Toddler Meaningful Auditory Integration Scale (IT-MAIS).At an assessment on October 2014, at 4 years of age, during a hearing perception testwith no visual cues, he attained the results shown in Cases of external auditory canal malformation or atresia often remain without adequatehearing rehabilitation, or end up being rehabilitated in an unsatisfactory manner, withthe use of bone vibration arches. These arches, the most often used way to providerehabilitation in these cases, are frequently poorly accepted by the user, due toesthetic reasons or mainly due to excessive compression on the skin. In addition to thestructural issue of this equipment, they have older and limited audiological technology,promoting hearing gains that are often ineffective.The first models of BAHA became commercially available abroad on 1987.In the studied case, due to the child's young age, we chose to use the equipment in itssoftband format, which is an option for using the same device with an elastic band. Asthe child grows and skull thickness consequently increases, the same equipment can beused, but attached to a titanium implant that is surgically inserted into the skullbone. Generally, the adequate bone thickness to receive this implant should beapproximately 5 mm, which is the thickness attained at around 5 years of age.,The rehabilitated individual shows excellent adaptation to the current method, bothregarding the acceptability of the device and the audiological gains provided by it. Themeasurement of these audiological gains provided by BAHA use through objective auditorytests is not feasible, due to the child's young age - hence the performance ofbehavioral tests and speech and hearing capacity protocols.,-We consider this report to be important, as there are few studies in our country usingthis technology in hearing rehabilitation. In addition, as far as the literature searchdemonstrated, no study in our language on the use of such equipment, specifically forauditory rehabilitation in Treacher Collins syndrome, was identified. In theinternational literature, the studies are not very numerous either.As our final considerations, we reaffirm the importance of disseminating knowledge aboutthe use of this equipment, as it is a new and effective alternative to auditoryrehabilitation. Patients with Treacher Collins syndrome comprise a group of individualsthat can largely benefit from the use of this technology. A síndrome de Treacher Collins, descrita pela primeira vez em 1900 pelooftalmologista britânico Edward Treacher Collins, é um distúrbio autossômicodominante que afeta um a cada 50.000 nascidos vivos.TCOF1 (locus 5q32),responsável por codificar a fosfoproteína nucleolar Treacle, queestá diretamente envolvida com o desenvolvimento dos dois primeiros arcosfaríngeos.,Nessa síndrome há uma mutação no gene locus 5q3,responsO fenótipo dos pacientes acometidos é variado. Há casos em que o paciente é levementeafetado e pode haver uma dificuldade de estabelecimento do diagnóstico; por outrolado, alguns pacientes têm morte precoce, no período perinatal, causada geralmentepelo grande comprometimento das vias aéreas.Entre 30% e 50% das crianças acometidas apresentam perda auditiva condutiva bilateralde grau severo devido a estenose ou atresia do conduto auditivo externo ou pormalformações da orelha média.Já as malformações do conduto auditivo externo fazem com que o uso de aparelhosauditivos tradicionais, os aparelhos de amplificação sonora individuais (AASI),raramente possam ser indicados. A opção de reabilitação mais comum é prótese auditivade vibração óssea, acoplada a um arco. Essa prótese apresenta uma série deinconvenientes, principalmente relacionados à pressão excessiva do arco, à estéticaruim e à dificuldade de se manter o arco em crianças, por ser facilmente removível.7Uma opção recente para reabilitação auditiva desses pacientes é a prótese auditivaancorada no osso (PAAO), que consiste em uma opção em casos de perdas condutivas oumistas que apresenta um baixo índice de complicações associado a bons resultadosfuncionais.O presente estudo relata o uso de PAAO na reabilitação auditiva de uma criançaportadora da síndrome de Treacher Collins, com atresia bilateral de condutosauditivos externos. Descrevemos também os métodos de avaliação auditiva e demensuração de resultados da reabilitação, para pacientes com essa síndrome e nessafaixa etária.Relato de caso baseado em revisão de prontuário aprovado pelo comitê de ética dainstituição sob o número 24692213.7.0000.0103 e com Termo de Consentimento Livre eEsclarecido assinado pela responsável pelo paciente.Paciente nascido em 23/09/10, masculino, branco, natural e residente em Curitiba, PR.Diagnosticado como portador da síndrome de Treacher Collins ao nascimento.Apresentava inclinação antimongoloide das fissuras palpebrais, hipoplasia malar,micrognatia e macrostomia. Contava ainda com microtia e atresia de conduto auditivoexterno bilateralmente.As complicações mais graves relacionadas à síndrome eram as das vias aéreas altas eaudição. Foi feita traqueostomia logo após o nascimento e gastrostomia aos trêsmeses. Cirurgias ortognáticas foram feitas com um ano e três meses, um ano e oitomeses e aos três anos e 10 meses. Atualmente, encontra-se estável e com boa adaptaçãono que diz respeito às vias respiratórias. Além disso, blefaroplastias foram feitasaos seis meses, oito meses, um ano e oito meses e um ano e 10 meses .Na avaliação auditiva feita em 25/04/12, com um ano e sete meses, foram aplicadostestes objetivos e subjetivos, conforme descritos a seguir:Potencial evocado auditivo de tronco encefálico: a criança apresentou limiaresde via aérea ausentes em 90dBA e limiares de via óssea presentes em 35dBA emambos os lados;Observação comportamental: esse teste fornece pistas e informações sobre odesenvolvimento global e principalmente o comportamento auditivo esperado parafaixa etária da criança, além de auxiliar no diagnóstico da deficiênciaauditiva e de outros distúrbios associados.softband, foiativado e adaptado. A observação comportamental registrou ganho funcional de 60dB e Infant-Toddler Meaningful Auditory Integration Scale(IT-Mais).Numa avaliação em 01/10/14, aos quatro anos, num teste de percepção auditiva sempista visual, obteve-se o resultado demonstrado na Casos de malformação ou atresia do conduto auditivo externo muitas vezes permanecemsem reabilitação auditiva adequada ou acabam reabilitados de maneira poucosatisfatória com o uso de arcos de vibração óssea. Esses arcos, a maneira dereabilitação mais usada nesses casos, muitas vezes são pouco aceitos pelo usuário porquestões estéticas ou, principalmente, por compressão excessiva sobre a pele. Além daquestão estrutural desses equipamentos, eles apresentam tecnologia audiológica maisantiga e limitada e promovem ganhos audiológicos muitas vezes pouco efetivos.Os primeiros modelos de PAAO passaram a ser comercialmente disponíveis no exterior em1987.softband, que é uma opção de uso do mesmo aparelho pormeio de uma faixa elástica. Com o crescimento da criança e consequentemente aumentoda espessura da calota craniana, o mesmo equipamento pode ser usado, mas acoplado aum implante de titânio cirurgicamente inserido no osso do crânio. Geralmente, aespessura adequada do osso para receber esse implante deve ser de aproximadamente 5mm, espessura essa que é adquirida por volta dos cinco anos.softband.No caso em questão, pela baixa idade da criança optou-se pelo uso do equipamento noseu formato ,O indivíduo reabilitado apresenta excelente adaptação ao método atual, tanto naaceitabilidade do dispositivo quanto dos ganhos audiológicos por ele propiciados. Amensuração desses ganhos audiológicos por meio de testes auditivos objetivos em usoda PAAO não são viáveis pela baixa idade da criança, daí a aplicação de testescomportamentais e protocolos de habilidade de fala e audição.,-Vemos como importante este relato pelo fato de ainda haver poucos estudos, em línguaportuguesa, com o uso dessa tecnologia na reabilitação auditiva. Além disso, até ondese estendeu o levantamento bibliográfico, nenhum estudo na nossa língua foiencontrado com o uso desse equipamento especificamente na reabilitação auditiva nasíndrome de Treacher Collins. Na literatura internacional eles também não são muitonumerosos.Como considerações finais, reafirma-se a importância da difusão do conhecimento sobreo uso desse equipamento, por ser ele uma nova e eficaz opção de reabilitaçãoauditiva. Pacientes portadores da síndrome de Treacher Collins se constituem numgrupo que pode ser muito beneficiado com o uso dessa tecnologia.