Exposure of iPSC-derived human microglia to brain substrates enables the generation and manipulation of diverse transcriptional states in vitro.

Microglia, the macrophages of the brain parenchyma, are key players in neurodegenerative diseases such as Alzheimer's disease. These cells adopt distinct transcriptional subtypes known as states. Understanding state function, especially in human microglia, has been elusive owing to a lack of tools to model and manipulate these cells. Here, we developed a platform for modeling human microglia transcriptional states in vitro. We found that exposure of human stem-cell-differentiated microglia to synaptosomes, myelin debris, apoptotic neurons or synthetic amyloid-beta fibrils generated transcriptional diversity that mapped to gene signatures identified in human brain microglia, including disease-associated microglia, a state enriched in neurodegenerative diseases. Using a new lentiviral approach, we demonstrated that the transcription factor MITF drives a disease-associated transcriptional signature and a highly phagocytic state. Together, these tools enable the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

Peripapillary and macular morpho-vascular changes in patients with genetic or clinical diagnosis of autosomal dominant optic atrophy: a case-control study.

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.

PERIPAPILLARY NEUROVASCULAR COUPLING IN THE EARLY STAGES OF DIABETIC RETINOPATHY.

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: ß = -2.95, P < 0.001; mild NPDR: ß = -1.76, P = 0.017; and moderate NPDR: ß = -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.

Ultrafast tissue staining with chemical tags.

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.

Cell types and neuronal circuitry underlying female aggression in Drosophila.

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 adult Drosophila melanogaster central 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.

Comparison of seating, powered characteristics and functions and costs of electrically powered wheelchairs in a general population of users.

PURPOSE: To profile and compare the seating and powered characteristics and functions of electrically powered wheelchairs (EPWs) in a general user population including equipment costs. METHOD: 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. RESULTS: 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. CONCLUSIONS: 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.

Functional and anatomical specificity in a higher olfactory centre.

Most sensory systems are organized into parallel neuronal pathways that process distinct aspects of incoming stimuli. In the insect olfactory system, second order projection neurons target both the mushroom body, required for learning, and the lateral horn (LH), proposed to mediate innate olfactory behavior. Mushroom body neurons form a sparse olfactory population code, which is not stereotyped across animals. In contrast, odor coding in the LH remains poorly understood. We combine genetic driver lines, anatomical and functional criteria to show that the Drosophila LH has ~1400 neurons and >165 cell types. Genetically labeled LHNs have stereotyped odor responses across animals and on average respond to three times more odors than single projection neurons. LHNs are better odor categorizers than projection neurons, likely due to stereotyped pooling of related inputs. Our results reveal some of the principles by which a higher processing area can extract innate behavioral significance from sensory stimuli.

Neurogenetic dissection of the Drosophila lateral horn reveals major outputs, diverse behavioural functions, and interactions with the mushroom body.

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.

Communication from Learned to Innate Olfactory Processing Centers Is Required for Memory Retrieval in Drosophila.

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.

Control devices for electrically powered wheelchairs: prevalence, defining characteristics and user perspectives.

PURPOSE: To determine the prevalence of control devices for electrically powered wheelchairs (EPWs), related characteristic features and users' views on their utility. METHOD: Postal survey of users of a regional NHS wheelchair service using a purpose-designed questionnaire (n = 262, ≥18 years old). RESULTS: 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. CONCLUSIONS: 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.

Facilitating Neuron-Specific Genetic Manipulations in Drosophila melanogaster Using a Split GAL4 Repressor.

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.

Insect Olfaction: Telling Food from Foe.

The 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.

Patient and equipment profile for wheelchair seating clinic provision.

PURPOSE: To characterise the provision of wheelchair seating both pre- and post-clinical intervention and compare and contrast the two largest diagnostic groups. METHOD: The case notes of those attending a wheelchair seating clinic for adults over a defined period were reviewed retrospectively. A classification system was devised that delineates between the complexity and type of equipment to gain a better understanding of provision. RESULTS: 146 patients were included; mean age 45 years (SD 16); 53.4% male. The two most prevalent primary medical diagnoses were cerebral palsy (CP) and multiple sclerosis (MS); 48.6% and 20.5%, respectively. The MS group, in comparison to the CP group, were significantly more likely to be older, new to seating provision, have been seen more recently, have a powered wheelchair, self-propel their manual wheelchair, have low rather than high complexity equipment and have their equipment changed following assessment. CONCLUSIONS: The equipment classification system will allow results from different studies to be readily compared. The results for those with CP and MS reflect the respective stable and progressive nature of these conditions. Referrals for those with MS should be prioritised. Wheelchair seating users with MS should be reassessed ∼18 months after provision. IMPLICATIONS FOR REHABILITATION: A detailed classification of wheelchair seating equipment based on a recognised standard vocabulary, such as the one proposed, is required to gain a better understanding of provision. Wheelchair seating equipment budget and staffing levels should reflect the diagnostic make up of a service's patient population. Referrals for people with MS should be prioritised as their current wheelchair seating provision is more likely not to be meeting their needs. People with MS should have a clinical review 18 months after wheelchair seating provision.

Clinical standards for National Health Service wheelchair and seating services in Scotland.

PURPOSE: To develop rigorous clinical standards for National Health Service (NHS) wheelchair and seating services in Scotland. METHOD: Clinical standards and an evaluation tool were developed by a working group using a well-established methodology. The available evidence was reviewed and a person-centre, iterative, consensus decision-making approach was employed to draft the standards. A public consultation was undertaken. The draft evaluation tool was tested during pilot visits to two wheelchair and seating centres. RESULTS: The majority of the 34 consultation responses were supportive, recognising the need to encourage and measure improvements and for a consistent approach to service delivery. Piloting found that the standards were challenging but achievable. The finalised standards and evaluation tool have been issued to health boards in Scotland. CONCLUSIONS: Following a structured, interdisciplinary and consultative process, the first clinical standards for NHS wheelchair and seating services in the Scotland were developed. They emphasise the need for an anticipatory approach, shifting from a reactive model of service delivery to a proactive one. Although developed in the Scottish policy context and service delivery model, many aspects of the standards will be applicable to services based in other parts of the UK and beyond. Implications for Rehabilitation Clinical standards should be developed and validated using established, rigorous methodology with a person-centre and consensus decision-making approach that includes public consultation and piloting. Wheelchair and seating service provision should be anticipatory and person-centre. The clinical standards and the supporting evaluation tool provide the basis for consistent, high-quality services for those individuals who require wheelchair and seating provision.