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In case-control single-cell RNA-seq studies, sample-level labels are transferred onto individual cells, labeling all case cells as affected, but only a small fraction of them may actually be perturbed. Here, using simulations, we demonstrate that the standard approach to single cell analysis fails to isolate the subset of affected case cells and their markers when either the affected subset is small, or when the strength of the perturbation is mild. To address this fundamental limitation, we introduce HiDDEN, a computational method that refines the case-control labels to accurately reflect the perturbation status of each cell. We show HiDDEN’s superior ability to recover biological signals missed by the standard analysis workflow in simulated ground truth datasets of cell type mixtures. When applied to a dataset of human multiple myeloma precursor conditions, HiDDEN recapitulates the expert manual annotation and discovers malignancy in previously considered healthy early stage samples. When applied to a mouse model of demyelination, HiDDEN identifies an endothelial subpopulation playing a role in early stage blood-brain barrier dysfunction. We anticipate that HiDDEN should find a wide usage in contexts which require the detection of subtle changes in cell types across conditions.

Microglia have emerged as key players in the pathogenesis of neurodegenerative conditions such as Alzheimer’s disease (AD). In response to CNS stimuli, these cells adopt distinct transcriptional and functional subtypes known as states. However, an understanding of the function of these states has been elusive, especially in human microglia, due to lack of tools to model and manipulate this cell-type. Here, we provide a platform for modeling human microglia transcriptional states in vitro. Using single-cell RNA sequencing, we found that exposure of human stem-cell differentiated microglia (iMGLs) to brain-related challenges generated extensive transcriptional diversity which mapped to gene signatures identified in human brain microglia. We identified two in vitro transcriptional clusters that were analogous to human and mouse disease-associated microglia (DAMs), a state enriched in neurodegenerative disease contexts. To facilitate scalable functional analyses, we established a lentiviral approach enabling broad and highly efficient genetic transduction of microglia in vitro. Using this new technology, we demonstrated that MITF (Melanocyte Inducing Transcription Factor), an AD-enriched transcription factor in microglia, drives both a disease-associated transcriptional signature and a highly phagocytic state. Finally, we confirmed these results across iMGLs differentiated from multiple iPSC lines demonstrating the broad utility of this platform. Together, these tools provide a comprehensive resource that enables the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

Aggressive social interactions are used to compete for limited resources and are regulated by complex sensory cues and the organism’s internal state. While both sexes exhibit aggression, its neuronal underpinnings are understudied in females. Here, we identify a population of sexually dimorphic aIPg neurons in the adultDrosophila melanogastercentral brain whose optogenetic activation increased, and genetic inactivation reduced, female aggression. Analysis of GAL4 lines identified in an unbiased screen for increased female chasing behavior revealed the involvement of another sexually dimorphic neuron, pC1d, and implicated aIPg and pC1d neurons as core nodes regulating female aggression. Connectomic analysis demonstrated that aIPg neurons and pC1d are interconnected and suggest that aIPg neurons may exert part of their effect by gating the flow of visual information to descending neurons. Our work reveals important regulatory components of the neuronal circuitry that underlies female aggressive social interactions and provides tools for their manipulation.

Aggressive social interactions are used to compete for limited resources and are regulated by complex sensory cues and the organism’s internal state. While both sexes exhibit aggression, its neuronal underpinnings are understudied in females. Here, we describe a set of connected neurons in the adultDrosophila melanogastercentral brain that drive female aggression. We identified a population of sexually dimorphic aIPg neurons whose optogenetic activation increased, and genetic inactivation reduced, female aggression. Analysis of GAL4 lines identified in an unbiased screen for increased female chasing behavior revealed the involvement of another sexually dimorphic neuron, pC1d, and implicated pC1d and aIPg neurons as core nodes regulating female aggression. pC1d activation increased female aggression and electron microscopy (EM) connectomic analysis demonstrated that aIPg neurons and pC1d have strong reciprocal connections. Our work reveals important regulatory components of the neuronal circuitry that underlies female aggressive social interactions and provides tools for their manipulation.

To profile and compare the seating and powered characteristics and functions of electrically powered wheelchairs (EPWs) in a general user population including equipment costs.Case notes of adult EPW users of a regional NHS service were reviewed retrospectively. Seating equipment complexity and type were categorized using the Edinburgh classification. Powered characteristics and functions, including control device type, were recorded.482 cases were included; 53.9% female; mean duration EPW use 8.1 years (SD 7.4); rear wheel drive 88.0%; hand joystick 94.8%. Seating complexity: low 73.2%, medium 18.0%, high 8.7%. Most prevalent diagnoses: multiple sclerosis (MS) 25.3%, cerebral palsy (CP) 18.7%, muscular dystrophy (8.5%). Compared to CP users, MS users were significantly older at first use, less experienced, more likely to have mid-wheel drive and less complex seating. Additional costs for muscular dystrophy and spinal cord injury users were 3-4 times stroke users.This is the first large study of a general EPW user population using a seating classification. Significant differences were found between diagnostic groups; nevertheless, there was also high diversity within each group. The differences in provision and the equipment costs across diagnostic groups can be used to improve service planning. Implications for Rehabilitation At a service planning level, knowledge of a population's diagnostic group and age distribution can be used to inform decisions about the number of required EPWs and equipment costs. At a user level, purchasing decisions about powered characteristics and functions of EPWs and specialised seating equipment need to be taken on a case by case basis because of the diversity of users' needs within diagnostic groups. The additional equipment costs for SCI and MD users are several times those of stroke users and add between 60 and 70% of the cost of basic provision.

Animals exhibit innate behaviours to a variety of sensory stimuli including olfactory cues. In Drosophila, one higher olfactory centre, the lateral horn (LH), is implicated in innate behaviour. However, our structural and functional understanding of the LH is scant, in large part due to a lack of sparse neurogenetic tools for this region. We generate a collection of split-GAL4 driver lines providing genetic access to 82 LH cell types. We use these to create an anatomical and neurotransmitter map of the LH and link this to EM connectomics data. We find ~30% of LH projections converge with outputs from the mushroom body, site of olfactory learning and memory. Using optogenetic activation, we identify LH cell types that drive changes in valence behavior or specific locomotor programs. In summary, we have generated a resource for manipulating and mapping LH neurons, providing new insights into the circuit basis of innate and learned olfactory behavior.

© Springer-Verlag GmbH Germany, part of Springer Nature 2019., Purpose: To evaluate the macular and peripapillary morpho-vascular changes in ADOA, using optical coherence tomography (OCT) and OCT angiography (OCTA). Methods: Prospectively defined, cross-sectional case-control study. Consecutive patients with a genetic or clinical diagnosis of ADOA along with age- and sex-matched controls were included. The radial peripapillary capillary (RPC) density and vessel density (VD) in the parafoveal superficial and deep capillary plexuses (SCP and DCP, respectively) were evaluated with OCTA. The ganglion cell complex (GCC) and retinal nerve fiber layer (RNFL) thickness were determined using structural OCT. We applied a previously validated customized macro (Fiji, SciJava Consortium) to compute RPC density. The remaining parameters were calculated by the built-in software. Non-parametric methods were used for data analysis. The target α level was 0.05, which was adjusted through Bonferroni's correction when multiple outcomes were tested. Results: Fifty-eight eyes (n = 29 control; n = 29 ADOA) from 30 subjects (mean age 42.43 ± 15.30 years; 37.93% male) were included. Parafoveal SCP VD, GCC thickness, RPC VD in the temporal quadrant, as well as RNFL thickness in the nasal and temporal quadrants were decreased in ADOA eyes (all p < 0.001). When only patients with genetically confirmed diagnosis were included, capillary dropout in the circumpapillary superior and inferior quadrants also became evident (both p < 0.001). The GCC/parafoveal SCP ratio was increased in ADOA, relatively to matched controls. In contrast, none of the circumpapillary morpho-vascular ratios was significantly different in ADOA eyes. Conclusions: The microvascular and structural changes found in ADOA suggest that both the macular and peripapillary regions are involved, although the threshold for damage of the structural and vascular components may be different for each region. Larger series with longitudinal follow-up may validate OCTA biomarkers helpful for disease monitoring.

Copyright © 2019, Wolters Kluwer Health. Ophthalmic Communications Society, Inc., Purpose: To study radial peripapillary capillary (RPC) density in the early stages of diabetic retinopathy (DR), using optical coherence tomography angiography. Methods: A cross-sectional evaluation of RPCs was performed using optical coherence tomography angiography (Avanti RTVue-XR 100, Optovue Inc, Fremont, CA). Annular RPC density was the primary outcome. Global density and retinal nerve fiber layer thickness were secondary outcomes. Diabetic eyes were divided into three groups: no DR, mild nonproliferative DR (mild NPDR), and moderate NPDR. Multilevel mixed-effects univariate and multivariate linear regression models were used. Results: We included 155 eyes (n = 42 control; n = 27 no DR; n = 28 mild NPDR; and n = 58 moderate NPDR) from 86 subjects (mean [SD] age 63.39 [10.70] years; 46.45% male). When compared with controls, a significant decrease in annular RPC density was found in all groups of diabetic eyes on multivariate analysis (no DR: β = -2.95, P < 0.001; mild NPDR: β = -1.76, P = 0.017; and moderate NPDR: β = -2.82, P < 0.001). We also detected a significant decrease in retinal nerve fiber layer thickness in diabetic eyes (even in the no DR group). Furthermore, in diabetic eyes, annular RPC density and retinal nerve fiber layer thickness correlated significantly (R = 0.4874, P < 0.001). Conclusion: Peripapillary neurovascular changes occur early in the course of DR. Their significance in the progression of DR warrants further research.

Animals exhibit innate behaviours in response to a variety of sensory stimuli such as olfactory cues. In Drosophila, a higher olfactory centre called the lateral horn (LH) is implicated in innate behaviour. However, our knowledge of the structure and function of the LH is scant, due to the lack of sparse neurogenetic tools for this brain region. Here we generate a collection of split-GAL4 driver lines providing genetic access to 82 LH cell-types. We identify the neurotransmitter and axo-dendritic polarity for each cell-type. Using these lines were create an anatomical map of the LH. We found that ∼30% of LH projections converge with outputs from the mushroom body, the site of olfactory learning and memory. Finally, using optogenetic activation of small groups of LH neurons. We identify cell-types that drive changes in either valence or specific motor programs, such as turning and locomotion. In summary we have generated a resource for manipulating and mapping LH neurons in both light and electron microscopy and generated insights into the anatomy and function of the LH.

Evolution has shaped nervous systems to produce stereotyped behavioural responses to ethologically relevant stimuli. For example when laying eggs, female Drosophila avoid geosmin, an odorant produced by toxic moulds. Here we identify second, third, and fourth order neurons required for this innate olfactory aversion. Connectomics data place these neurons in a complete synaptic circuit from sensory input to descending output. We find multiple levels of valence-specific convergence, including a novel form of axo-axonic input onto second order neurons conveying another danger signal, the pheromone of parasitoid wasps. However, we also observe extensive divergence: second order geosmin neurons connect with a diverse array of 80 third order cell types. We find a pattern of convergence of aversive odour channels at this level. Crossing one more synaptic layer, we identified descending neurons critical for egg-laying aversion. Our data suggest a transition from a labelled line organisation in the periphery to a highly distributed central brain representation that is then coupled to distinct descending pathways.

Most sensory systems are organized into parallel neuronal pathways that process distinct aspects of incoming stimuli. For example, in insects, second order olfactory projection neurons target both the mushroom body, which is required for learning, and the lateral horn (LH), which has been proposed to mediate innate olfactory behavior. Mushroom body neurons encode odors in a sparse population code, which does not appear stereotyped across animals. In contrast the functional principles of odor coding in the LH remain poorly understood. We have carried out a comprehensive anatomical analysis of the Drosophila LH, counting ~1400 neurons; combining genetic driver lines, anatomical and functional criteria, we identify 165 LHN cell types. We then show that genetically labeled LHNs have stereotyped odor responses across animals for 33 of these cell types. LHN tuning can be ultra-sparse (1/40 odors tested), but on average single LHNs respond to three times more odors than single projection neurons. This difference can be rationalized by our observation that LHNs are better odor categorizers, likely due to pooling of input projection neurons responding to different odors of the same category. Our results reveal some of the principles by which a higher sensory processing area can extract innate behavioral significance from sensory stimuli.

SummaryThe same sensory signal can be interpreted differently according to context. A new study in Drosophila uses cell-type-specific tools to identify neural circuits that integrate context during olfactory processing and surprisingly implicates memory-recall neurons.

Summary The behavioral response to a sensory stimulus may depend on both learned and innate neuronal representations. How these circuits interact to produce appropriate behavior is unknown. In Drosophila, the lateral horn (LH) and mushroom body (MB) are thought to mediate innate and learned olfactory behavior, respectively, although LH function has not been tested directly. Here we identify two LH cell types (PD2a1 and PD2b1) that receive input from an MB output neuron required for recall of aversive olfactory memories. These neurons are required for aversive memory retrieval and modulated by training. Connectomics data demonstrate that PD2a1 and PD2b1 neurons also receive direct input from food odor-encoding neurons. Consistent with this, PD2a1 and PD2b1 are also necessary for unlearned attraction to some odors, indicating that these neurons have a dual behavioral role. This provides a circuit mechanism by which learned and innate olfactory information can interact in identified neurons to produce appropriate behavior. Video Abstract, Highlights • Specific Drosophila lateral horn neurons mediate innate attraction to food odors • The same neurons receive plastic odor information from the mushroom body • Recall after associative learning depends on reduced drive to lateral horn neurons • Connectomics circuit for integration of learned and innate odor representations, Sensory stimuli can engage both learned and innate behaviors. Dolan et al. identify neurons in Drosophila that directly integrate unlearned and plastic odor representations; they are required for innate approach to food odors but also learned aversive recall.

Many animals can show either learned or innate behavioural responses to a given stimulus. However, how innate and memory circuits interact to produce an appropriate behavioural response is unknown. The Drosophila olfactory system is an excellent model to study how sensory stimuli are processed, stored and transformed into behaviour. Its numerical simplicity, powerful genetics and similarity to mammalian olfactory circuits have enabled many insights into sensory processing and memory. Two higher brain regions, the lateral horn (LH) and the mushroom body (MB) are thought to mediate innate and learned olfactory behaviour respectively, although the function of the LH has not been directly tested. Here we identify a LH cell-type (PD2a1/b1) that receives input from the MB. In contrast to the above model that LH is only necessary for innate behaviour, this cell-type is required for memory retrieval. Moreover, the activity of this cholinergic cell-type is modulated by training, indicating that memory information is passed to the innate olfactory processing centre. PD2a1/b1 dendrites likely receive input from appetitive olfactory projection neurons while their axons project to convergence zones of the MB extrinsic neurons and interdigitate with MB input and output neurons. This work demonstrates that LH neurons play a role in memory retrieval and illustrates the extensive interconnectivity of these two higher brain regions.

Wheelchairs are, for users, a primary means of mobility and an important means of performing activities of daily living. A common, accepted vocabulary is required to support and foster evidence-based practice and communication amongst professionals and with users. The international standard for wheelchair vocabulary, ISO 7176-26:2007, specifies terms and definitions with the purpose of eliminating confusion from the duplication or inappropriate use of terms. The aim of this study was to assess its impact and, based on that assessment, critically appraise the standard. Two databases were searched returning 189 and 283 unique articles with wheelchair in the title published between 2004–2006 and 2009–2011 respectively. Compliance, based on title and abstract usage, was poor, ranging from 0 to 50% correct usage, with no significant difference between pre- and post-publication. A review of prescription forms found only 9% correct usage. A survey of NHS wheelchair managers found that only 30% were positive that they had a copy despite 67% agreeing that the standard is important. The ISO wheelchair vocabulary standard was found not to be achieving its stated purpose. It is recommended that it be revised taking into account the findings of this study including the need for targeted dissemination and increased awareness.

Efforts to map neural circuits have been galvanized by the development of genetic technologies that permit the manipulation of targeted sets of neurons in the brains of freely behaving animals. The success of these efforts relies on the experimenter’s ability to target arbitrarily small subsets of neurons for manipulation, but such specificity of targeting cannot routinely be achieved using existing methods. In Drosophila melanogaster, a widely-used technique for refined cell type-specific manipulation is the Split GAL4 system, which augments the targeting specificity of the binary GAL4-UAS (Upstream Activating Sequence) system by making GAL4 transcriptional activity contingent upon two enhancers, rather than one. To permit more refined targeting, we introduce here the “Killer Zipper” (KZip+), a suppressor that makes Split GAL4 targeting contingent upon a third enhancer. KZip+ acts by disrupting both the formation and activity of Split GAL4 heterodimers, and we show how this added layer of control can be used to selectively remove unwanted cells from a Split GAL4 expression pattern or to subtract neurons of interest from a pattern to determine their requirement in generating a given phenotype. To facilitate application of the KZip+ technology, we have developed a versatile set of LexAop-KZip+ fly lines that can be used directly with the large number of LexA driver lines with known expression patterns. KZip+ significantly sharpens the precision of neuronal genetic control available in Drosophila and may be extended to other organisms where Split GAL4-like systems are used.

To determine the prevalence of control devices for electrically powered wheelchairs (EPWs), related characteristic features and users' views on their utility.Postal survey of users of a regional NHS wheelchair service using a purpose-designed questionnaire (n = 262, ≥18 years old).Mean age 54.4 years, female 56.8%, mean duration EPW use 10.1 years, mean usage 6.7 days per week and 9.2 h per day. Largest diagnostic groups: Multiple Sclerosis 28.3%, Cerebral Palsy 13.8% and Spinal Cord Injury 11.7%. Control device types 94.6% hand joystick, 2.3% chin joystick, 2.7% switches and 0.4% foot control. 42.4% reported fatigue or tiredness and 38.8% pain or discomfort limited EPW use. 28.0% reported an accident or mishap.This is the first study of control devices on a large, general population of EPW users. The majority have control devices that meet their needs, with high levels of user satisfaction, though some might benefit from adjustments or modifications to their current provision and others might benefit by changing to a different type of control device. High proportions reported fatigue or tiredness and pain or discomfort limit their EPW use. The study provides indicators for prescribers and manufacturers of control devices for EPWs. Implications for Rehabilitation Most users have control devices that meet their needs, with high levels of satisfaction, but some would benefit from adjustments or modifications or a change of type. A high proportion reported fatigue or tiredness and pain or discomfort limit their use of their EPW and prescribers need to be mindful of these issues when determining the most suitable type of control device and where it should be positioned. The vast majority of users have a hand joystick as a control device with alternative control devices (such as chin joysticks and switches) being far less prevalent. Adverse incidents may arise due to difficulty with manoeuvring or accidental activation of the hand joystick that can lead to collisions and even entrapment.

Genetically encoded fluorescent proteins and immunostainings are widely used to detect cellular or sub-cellular structures in thick biological samples. However, each approach suffers from limitations, including low signal and limited spectral flexibility or slow speed, poor penetration and high background, respectively. Here we overcome these limitations by using transgenically expressed chemical tags for rapid, even and low-background labeling of thick biological tissues. We construct a platform of widely applicable transgenic Drosophila reporter lines, demonstrating that chemical labeling can accelerate staining of whole-mount fly brains by a factor of 100x. Together, this tag-based approach drastically improves the speed and specificity of labeling genetically marked cells in intact and/or thick biological samples.

A low-level, closed-loop controller for FES-assisted standing up and sitting down is described. If, for able-bodied individuals, when standing up and sitting down, the knee angular velocity is plotted against knee angle, consistent phase-plane trajectories are produced. The bang-bang controller uses a model of this trajectory as a switching curve. The design rationale for the controller was the desire to avoid injuries that might occur if knee-locking on standing up and seat-contact on sitting down are not adequately controlled. This switching curve controller (SCC) was incorporated within a hierarchical, finite state control scheme, with electrical stimulation applied bilaterally to the knee extensors. The SCC was tested in a pilot study on a female volunteer with paraplegia (T5/6 ASIA A) and evaluated against an unramped, open-loop controller (OLC). The vertical hand forces and knee angles were measured. The subject was able to achieve standing up and sitting down safely using both controllers. For standing up, the SCC was not found to offer any quantifiable advantages over the OLC and was found to increase the hand force by 8.4%. In contrast, for sitting down the SCC was found to reduce the knee angular velocities as the subject approached the seat by 27%, demonstrating a safer, softer landing.

Following work done in C.E.C. project AIM 2002 CAMARC II, a framework of the relevant standards for testing equipment used in laboratories analysing human movement is presented. In particular, gait analysis laboratories may provide a service for a clinical team considering treatment such as surgical intervention or provision of orthoses. In this case the analysis and equipment used in the analysis must have assured quality so that the correct clinical interpretation of the results is possible. The operation of a gait analysis laboratory may be described in quality standards terminology as the operation of a ‘special process’. The operation of a special process requires that particular attention be paid to procedures including confirmation of the equipment and regular testing to ensure correct operation of the equipment. In general this is difficult to achieve since equipment manufacturers still have to address this technical requirement. Although difficult, these procedures must be accomplished in the operation of a special process. Passages of the most pertinent standards are presented and a methodology of periodic confirmation and regular spot checking of equipment suggested to ensure its correct operation. Although force plates are specifically addressed, the framework could be applied to other dynamometers and measurement equipment.

An in situ calibration protocol for ground-to-foot force measuring platforms is described. The methodology allows verification of the function of the force plate and allows accurate calibration for three force and moment channels. The effect of cross-sensitivity on recorded data is discussed along with the need for improvements in methodology to quantify this property.

Efforts to map neural circuits have been galvanized by the development of genetic technologies that permit the manipulation of targeted sets of neurons in the brains of freely behaving animals. The success of these efforts relies on the experimenter’s ability to target arbitrarily small subsets of neurons for manipulation, but such specificity of targeting cannot routinely be achieved using existing methods. In Drosophila melanogaster, a widely-used technique for refined cell type-specific manipulation is the Split GAL4 system, which augments the targeting specificity of the binary GAL4-UAS (Upstream Activating Sequence) system by making GAL4 transcriptional activity contingent upon two enhancers, rather than one. To permit more refined targeting, we introduce here the “Killer Zipper” (KZip+), a suppressor that makes Split GAL4 targeting contingent upon a third enhancer. KZip+ acts by disrupting both the formation and activity of Split GAL4 heterodimers, and we show how this added layer of control can be used to selectively remove unwanted cells from a Split GAL4 expression pattern or to subtract neurons of interest from a pattern to determine their requirement in generating a given phenotype. To facilitate application of the KZip+ technology, we have developed a versatile set of LexAop-KZip+ fly lines that can be used directly with the large number of LexA driver lines with known expression patterns. KZip+ significantly sharpens the precision of neuronal genetic control available in Drosophila and may be extended to other organisms where Split GAL4-like systems are used.

Genetically encoded fluorescent proteins and immunostaining are widely used to detect cellular and subcellular structures in fixed biological samples. However, for thick or whole-mount tissue, each approach suffers from limitations, including limited spectral flexibility and lower signal or slow speed, poor penetration, and high background labeling, respectively. We have overcome these limitations by using transgenically expressed chemical tags for rapid, even, high-signal and low-background labeling of thick biological tissues. We first construct a platform of widely applicable transgenic Drosophila reporter lines, demonstrating that chemical labeling can accelerate staining of whole-mount fly brains by a factor of 100. Using viral vectors to deliver chemical tags into the mouse brain, we then demonstrate that this labeling strategy works well in mice. Thus this tag-based approach drastically improves the speed and specificity of labeling genetically marked cells in intact and/or thick biological samples.