Multilevel visuomotor control of locomotion in Drosophila.

Vision is critical for the control of locomotion, but the underlying neural mechanisms by which visuomotor circuits contribute to the movement of the body through space are yet not well understood. Locomotion engages multiple control systems, forming distinct interacting "control levels" driven by the activity of distributed and overlapping circuits. Therefore, a comprehensive understanding of the mechanisms underlying locomotion control requires the consideration of all control levels and their necessary coordination. Due to their small size and the wide availability of experimental tools, Drosophila has become an important model system to study this coordination. Traditionally, insect locomotion has been divided into studying either the biomechanics and local control of limbs, or navigation and course control. However, recent developments in tracking techniques, and physiological and genetic tools in Drosophila have prompted researchers to examine multilevel control coordination in flight and walking.

Circuits for self-motion estimation and walking control in Drosophila.

The brain's evolution and operation are inextricably linked to animal movement, and critical functions, such as motor control, spatial perception, and navigation, rely on precise knowledge of body movement. Such internal estimates of self-motion emerge from the integration of mechanosensory and visual feedback with motor-related signals. Thus, this internal representation likely depends on the activity of circuits distributed across the central nervous system. However, the circuits responsible for self-motion estimation, and the exact mechanisms by which motor-sensory coordination occurs within these circuits remain poorly understood. Recent technological advances have positioned Drosophila melanogaster as an advantageous model for investigating the emergence, maintenance, and utilization of self-motion representations during naturalistic walking behaviors. In this review, I will illustrate how the adult fly is providing insights into the fundamental problems of self-motion computations and walking control, which have relevance for all animals.

Motion vision: Drosophila neural pathways that go with the visual flow.

Processing visual motion cues to interpret self-motion, the movement of others, and the environment's structure is vital to all animals, whether prey or predator. A new study in Drosophila identifies multiple pathways likely contributing to visual motion-dependent computations and behaviors.

Walking strides direct rapid and flexible recruitment of visual circuits for course control in Drosophila.

Flexible mapping between activity in sensory systems and movement parameters is a hallmark of motor control. This flexibility depends on the continuous comparison of short-term postural dynamics and the longer-term goals of an animal, thereby necessitating neural mechanisms that can operate across multiple timescales. To understand how such body-brain interactions emerge across timescales to control movement, we performed whole-cell patch recordings from visual neurons involved in course control in Drosophila. We show that the activity of leg mechanosensory cells, propagating via specific ascending neurons, is critical for stride-by-stride steering adjustments driven by the visual circuit, and, at longer timescales, it provides information about the moving body's state to flexibly recruit the visual circuit for course control. Thus, our findings demonstrate the presence of an elegant stride-based mechanism operating at multiple timescales for context-dependent course control. We propose that this mechanism functions as a general basis for the adaptive control of locomotion.

Fast tuning of posture control by visual feedback underlies gaze stabilization in walking Drosophila.

Locomotion requires a balance between mechanical stability and movement flexibility to achieve behavioral goals despite noisy neuromuscular systems, but rarely is it considered how this balance is orchestrated. We combined virtual reality tools with quantitative analysis of behavior to examine how Drosophila uses self-generated visual information (reafferent visual feedback) to control gaze during exploratory walking. We found that flies execute distinct motor programs coordinated across the body to maximize gaze stability. However, the presence of inherent variability in leg placement relative to the body jeopardizes fine control of gaze due to posture-stabilizing adjustments that lead to unintended changes in course direction. Surprisingly, whereas visual feedback is dispensable for head-body coordination, we found that self-generated visual signals tune postural reflexes to rapidly prevent turns rather than to promote compensatory rotations, a long-standing idea for visually guided course control. Together, these findings support a model in which visual feedback orchestrates the interplay between posture and gaze stability in a manner that is both goal dependent and motor-context specific.

DeepEthogram, a machine learning pipeline for supervised behavior classification from raw pixels.

Videos of animal behavior are used to quantify researcher-defined behaviors of interest to study neural function, gene mutations, and pharmacological therapies. Behaviors of interest are often scored manually, which is time-consuming, limited to few behaviors, and variable across researchers. We created DeepEthogram: software that uses supervised machine learning to convert raw video pixels into an ethogram, the behaviors of interest present in each video frame. DeepEthogram is designed to be general-purpose and applicable across species, behaviors, and video-recording hardware. It uses convolutional neural networks to compute motion, extract features from motion and images, and classify features into behaviors. Behaviors are classified with above 90% accuracy on single frames in videos of mice and flies, matching expert-level human performance. DeepEthogram accurately predicts rare behaviors, requires little training data, and generalizes across subjects. A graphical interface allows beginning-to-end analysis without end-user programming. DeepEthogram's rapid, automatic, and reproducible labeling of researcher-defined behaviors of interest may accelerate and enhance supervised behavior analysis. Code is available at: https://github.com/jbohnslav/deepethogram.

A Single MicroRNA-Hox Gene Module Controls Equivalent Movements in Biomechanically Distinct Forms of Drosophila.

Movement is the main output of the nervous system. It emerges during development to become a highly coordinated physiological process essential to survival and adaptation of the organism to the environment. Similar movements can be observed in morphologically distinct developmental stages of an organism, but it is currently unclear whether or not these movements have a common molecular cellular basis. Here we explore this problem in Drosophila, focusing on the roles played by the microRNA (miRNA) locus miR-iab4/8, which we previously showed to be essential for the normal corrective response displayed by the fruit fly larva when turned upside down (self-righting). Our study shows that miR-iab4 is required for normal self-righting across all three Drosophila larval stages. Unexpectedly, we also discover that this miRNA is essential for normal self-righting behavior in the adult fly, an organism with different morphology, neural constitution, and biomechanics. Through the combination of gene expression, optical imaging, and quantitative behavioral approaches, we provide evidence that miR-iab4 exerts its effects on adult self-righting behavior in part through repression of the Hox gene Ultrabithorax (Ubx) in a specific set of adult motor neurons, the NB2-3/lin15 neurons. Our results show that miRNA controls the function, rather than the morphology, of these neurons and demonstrate that post-developmental changes in Hox gene expression can modulate behavior in the adult. Our work reveals that a common miRNA-Hox genetic module can be re-deployed in different neurons to control functionally equivalent movements in biomechanically distinct organisms and describes a novel post-developmental role of the Hox genes in adult neural function.

Sensation during Active Behaviors.

A substantial portion of our sensory experience happens during active behaviors such as walking around or paying attention. How do sensory systems work during such behaviors? Neural processing in sensory systems can be shaped by behavior in multiple ways ranging from a modulation of responsiveness or sharpening of tuning to a dynamic change of response properties or functional connectivity. Here, we review recent findings on the modulation of sensory processing during active behaviors in different systems: insect vision, rodent thalamus, and rodent sensory cortices. We discuss the circuit-level mechanisms that might lead to these modulations and their potential role in sensory function. Finally, we highlight the open questions and future perspectives of this exciting new field.

A faithful internal representation of walking movements in the Drosophila visual system.

The integration of sensorimotor signals to internally estimate self-movement is critical for spatial perception and motor control. However, which neural circuits accurately track body motion and how these circuits control movement remain unknown. We found that a population of Drosophila neurons that were sensitive to visual flow patterns typically generated during locomotion, the horizontal system (HS) cells, encoded unambiguous quantitative information about the fly's walking behavior independently of vision. Angular and translational velocity signals were integrated with a behavioral-state signal and generated direction-selective and speed-sensitive graded changes in the membrane potential of these non-spiking cells. The nonvisual direction selectivity of HS cells cooperated with their visual selectivity only when the visual input matched that expected from the fly's movements, thereby revealing a circuit for internally monitoring voluntary walking. Furthermore, given that HS cells promoted leg-based turning, the activity of these cells could be used to control forward walking.

Neural correlates of illusory motion perception in Drosophila.

When the contrast of an image flickers as it moves, humans perceive an illusory reversal in the direction of motion. This classic illusion, called reverse-phi motion, has been well-characterized using psychophysics, and several models have been proposed to account for its effects. Here, we show that Drosophila melanogaster also respond behaviorally to the reverse-phi illusion and that the illusion is present in dendritic calcium signals of motion-sensitive neurons in the fly lobula plate. These results closely match the predictions of the predominant model of fly motion detection. However, high flicker rates cause an inversion of the reverse-phi behavioral response that is also present in calcium signals of lobula plate tangential cell dendrites but not predicted by the model. The fly's behavioral and neural responses to the reverse-phi illusion reveal unexpected interactions between motion and flicker signals in the fly visual system and suggest that a similar correlation-based mechanism underlies visual motion detection across the animal kingdom.

Walking modulates speed sensitivity in Drosophila motion vision.

Changes in behavioral state modify neural activity in many systems. In some vertebrates such modulation has been observed and interpreted in the context of attention and sensorimotor coordinate transformations. Here we report state-dependent activity modulations during walking in a visual-motor pathway of Drosophila. We used two-photon imaging to monitor intracellular calcium activity in motion-sensitive lobula plate tangential cells (LPTCs) in head-fixed Drosophila walking on an air-supported ball. Cells of the horizontal system (HS)--a subgroup of LPTCs--showed stronger calcium transients in response to visual motion when flies were walking rather than resting. The amplified responses were also correlated with walking speed. Moreover, HS neurons showed a relatively higher gain in response strength at higher temporal frequencies, and their optimum temporal frequency was shifted toward higher motion speeds. Walking-dependent modulation of HS neurons in the Drosophila visual system may constitute a mechanism to facilitate processing of higher image speeds in behavioral contexts where these speeds of visual motion are relevant for course stabilization.

Two-photon calcium imaging from head-fixed Drosophila during optomotor walking behavior.

Drosophila melanogaster is a model organism rich in genetic tools to manipulate and identify neural circuits involved in specific behaviors. Here we present a technique for two-photon calcium imaging in the central brain of head-fixed Drosophila walking on an air-supported ball. The ball's motion is tracked at high resolution and can be treated as a proxy for the fly's own movements. We used the genetically encoded calcium sensor, GCaMP3.0, to record from important elements of the motion-processing pathway, the horizontal-system lobula plate tangential cells (LPTCs) in the fly optic lobe. We presented motion stimuli to the tethered fly and found that calcium transients in horizontal-system neurons correlated with robust optomotor behavior during walking. Our technique allows both behavior and physiology in identified neurons to be monitored in a genetic model organism with an extensive repertoire of walking behaviors.

Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.

Genetically encoded calcium indicators (GECIs) can be used to image activity in defined neuronal populations. However, current GECIs produce inferior signals compared to synthetic indicators and recording electrodes, precluding detection of low firing rates. We developed a single-wavelength GCaMP2-based GECI (GCaMP3), with increased baseline fluorescence (3-fold), increased dynamic range (3-fold) and higher affinity for calcium (1.3-fold). We detected GCaMP3 fluorescence changes triggered by single action potentials in pyramidal cell dendrites, with signal-to-noise ratio and photostability substantially better than those of GCaMP2, D3cpVenus and TN-XXL. In Caenorhabditis elegans chemosensory neurons and the Drosophila melanogaster antennal lobe, sensory stimulation-evoked fluorescence responses were significantly enhanced with GCaMP3 (4-6-fold). In somatosensory and motor cortical neurons in the intact mouse, GCaMP3 detected calcium transients with amplitudes linearly dependent on action potential number. Long-term imaging in the motor cortex of behaving mice revealed large fluorescence changes in imaged neurons over months.

The structural and functional differentiation of hair cells in a lizard's basilar papilla suggests an operational principle of amniote cochleas.

The hair cells in the mammalian cochlea are of two distinct types. Inner hair cells are responsible for transducing mechanical stimuli into electrical responses, which they forward to the brain through a copious afferent innervation. Outer hair cells, which are thought to mediate the active process that sensitizes and tunes the cochlea, possess a negligible afferent innervation. For every inner hair cell, there are approximately three outer hair cells, so only one-quarter of the hair cells directly deliver information to the CNS. Although this is a surprising feature for a sensory system, the occurrence of a similar innervation pattern in birds and crocodilians suggests that the arrangement has an adaptive value. Using a lizard with highly developed hearing, the tokay gecko, we demonstrate in the present study that the same principle operates in a third major group of terrestrial animals. We propose that the differentiation of hair cells into signaling and amplifying classes reflects incompatible strategies for the optimization of mechanoelectrical transduction and of an active process based on active hair-bundle motility.

Sexually transmitted and reproductive tract infections in symptomatic clients of pharmacies in Lima, Peru.

OBJECTIVE: To determine prevalences and predictors of sexually transmitted and reproductive tract infections among men and women seeking care at pharmacies. METHODS: Men and women with urethral discharge or dysuria and vaginal discharge were enrolled at 12 central and 52 smaller pharmacies in Lima, Peru. All participants answered a questionnaire. Men provided urine for polymerase chain reaction (PCR) testing for Neisseria gonorrhoeae and Chlamydia trachomatis, and for leucocyte esterase testing. Women provided self-obtained vaginal swabs for PCR testing for N gonorrhoeae and C trachomatis, Trichomonas vaginalis culture and bacterial vaginosis and Candida. RESULTS: Among 106 symptomatic men, N gonorrhoeae and C trachomatis were detected in 34% and were associated with urethral discharge compared with dysuria only (odds ratio (OR) 4.3, p = 0.003), positive urine leucocyte esterase testing (OR 7.4, p = 0.009), less education (OR 5.5, p = 0.03), and with symptoms for <5 days (OR 2.5, p = 0.03). Among 121 symptomatic women, 39% had bacterial vaginosis or T vaginalis, and 7.7% had candidiasis. N gonorrhoeae and C trachomatis were detected in 12.4% of the women. Overall, 48.8% had one or more of these infections. No factors were associated with vaginal infection, and only symptoms of vaginal discharge for <5 days were associated with N gonorrhoeae and C trachomatis (OR 4.0, p = 0.02). The main reason reported for seeking advice at pharmacies by both men and women was trust in pharmacy workers. CONCLUSIONS: Among men and women presenting to pharmacies with urethral and vaginal symptoms, rates of urethral and vaginal infections were comparable to those found in other clinical settings. Pharmacies can contribute to the care and prevention of sexually transmitted infection in developing countries.

Utility of the Determine Syphilis TP rapid test in commercial sex venues in Peru.

OBJECTIVES: This study sought to evaluate the utility of the Determine Syphilis TP test performed in Peruvian commercial sex venues for the detection of active syphilis; and determine the feasibility of integrating rapid syphilis testing for female sex workers (FSW) into existing health outreach services. METHODS: We tested 3586 female sex workers for syphilis by Determine in the field using whole blood fingerstick, and by rapid plasma reagin (RPR) and Treponema pallidum haemagglutination assay (TPHA) in a central laboratory in Lima using sera. RESULTS: 97.4% of the FSW offered rapid syphilis testing participated; and among those who tested positive, 87% visited the local health centre for treatment. More than twice as many specimens were RPR reactive using serum in Lima (5.7%) than tested positive by whole blood Determine in the field (2.8%), and although most were confirmed by TPHA, only a small proportion (0.7%) were RPR reactive at >or=1:8 dilutions, and likely indicating active syphilis. Sensitivity, specificity and positive predictive value of the Determine Syphilis TP test in whole blood when compared to serum RPR reactivity at any dilution confirmed by TPHA as the gold standard were 39.3%, 99.2% and 71.4%, respectively. Sensitivity improved to 64.0% when using serum RPR >or=1:8 confirmed by TPHA. Invalid tests were rare (0.3%). CONCLUSIONS: Rapid syphilis testing in sex work venues proved feasible, but Determine using whole blood obtained by fingerstick was substantially less sensitive than reported in previous laboratory-based studies using serum. Although easy to perform in outreach venues, the utility of this rapid syphilis test was relatively low in settings where a large proportion of the targeted population has been previously tested and treated.

Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction.

Fruit flies are attracted by a diversity of odors that signal the presence of food, potential mates, or attractive egg-laying sites. Most Drosophila olfactory neurons express two types of odorant receptor genes: Or83b, a broadly expressed receptor of unknown function, and one or more members of a family of 61 selectively expressed receptors. While the conventional odorant receptors are highly divergent, Or83b is remarkably conserved between insect species. Two models could account for Or83b function: it could interact with specific odor stimuli independent of conventional odorant receptors, or it could act in concert with these receptors to mediate responses to all odors. Our results support the second model. Dendritic localization of conventional odorant receptors is abolished in Or83b mutants. Consistent with this cellular defect, the Or83b mutation disrupts behavioral and electrophysiological responses to many odorants. Or83b therefore encodes an atypical odorant receptor that plays an essential general role in olfaction.

Expression of 3 beta-hydroxysteroid dehydrogenase in early bovine embryo development.

We analyzed the presence of 3 beta-Hydroxysteroid Dehydrogenase/Delta(5-->4)-isomerase enzyme (3 beta-HSD) activity, a key enzyme of the steroid metabolic pathway, the mRNA of this enzyme, and the steroid metabolism in in vitro produced bovine embryos. 3 beta-HSD activity was detected in in vitro matured oocytes (74.4 +/- 1.4%), 1-cell (72.9 +/- 6.1%), 2-cell (61.8 +/- 7.4%), 8-cell (50 +/- 5%), morulae (50.8 +/- 2.6%), blastocysts (94.4 +/- 3%), and hatched blastocysts (100 +/- 0%) meanwhile the 4-cell stage showed a significant reduction (16.7 +/- 4.7%). When total embryonic RNA of different stages was subjected to RT-PCR assays, the mRNA of 3 beta-HSD was found to be present in all developmental stages of in vitro produced bovine embryos, from the oocyte to the blastocyst, with a marked decrease at the 4-cell stage. To determine whether the temporal pattern of enzyme activity was dependent on the maternal to zygotic transition, embryos were incubated in the presence of a transcription inhibitor, alpha-amanitin. The reappearance of the enzyme activity after the 4-cell stage was blocked in alpha-amanitin treated embryos, indicating the requirement of embryonic transcription. On the other hand, the embryonic steroid metabolism was tested by incubating blastocyst with tritiated pregnenolone. Analysis of the metabolites by TLC indicated the production of a compound with a mobility identical to progesterone. These results described the expression of the 3 beta-HSD and the activity of this metabolic enzyme in bovine oocytes and preimplantation embryos, suggesting that steroids may act as autocrine effectors on preimplantation embryo development.

Analysis of biomechanical effects on bone and on the muscle-bone interactions in small animal models.

Animal models are suitable to study many aspects of bone structure and strength. This article reviews some general principles of current bone biomechanics and describes the scope of the available methodology for biomechanical studies of the musculoskeletal system employing those models. The analysis comprises bone and muscle "mass" indicators provided by standard densitometry (DEXA); bone 'mass', 'apparent density', geometry or architectural design and strength and muscle strength indicators that can be determined by peripheral quantitative computed tomography (pQCT), and bone material and structural (whole-bone) properties than can be directly assessed by destructive mechanical tests. Some novel interrelationships that can be investigated that way are discussed, namely, 1. the pathogenetic analysis of the effects on whole-bone strength, 2. the discrimination between mineralization and microstructural factors as determinants of changes in the bone material or structural properties, 3. the evaluation of the interaction of a treatment with the ability of bone 'mechanostat' to optimize the bone architectural design by 'distribution / mass' and 'distribution / quality' curves, and 4. the analysis of effects on the musclebone interactions for a differential diagnosis between 'physiological' or 'disuse' and 'true' osteopenias and osteoporoses.

Effects of calcipotriol on stratum corneum barrier function, hydration and cell renewal in humans.

Calcipotriol, a vitamin D analogue utilized for psoriasis, has irritation as its most frequent reported adverse event. However, studies on its irritant properties in humans have produced conflicting data. This study evaluates the effect of calcipotriol on stratum corneum barrier function, hydration and cell turnover in healthy volunteers, compared with sodium lauryl sulphate (SLS) as a model irritant. Calcipotriol 0.005% ointment and 1% aqueous SLS solution were applied for 60 min once daily for 2 weeks (5 consecutive days weekly) on untreated and on dansyl-chloride-labelled skin. Irritant responses were documented by visual scoring and by measurement of the transepidermal water loss (TEWL) and stratum corneum hydration (electrical capacitance), until day 18. Stratum corneum turnover time (SCTT) was the time in days between staining (day 0) and the disappearance of dansyl fluorescence. SLS caused more erythema, scaling, and a significant TEWL increase for 18 days. In contrast, calcipotriol induced erythema, and slightly but significantly increased TEWL on day 11 only, as compared with the vehicle control (P < 0.05). SLS, but not calcipotriol, caused skin dryness from day 4 to day 18. The shortest SCTT was obtained at SLS-exposed sites (11.2 +/- 0.7 days: mean +/- SD). Calcipotriol significantly shortened SCTT (16.3 +/- 1.1 days) when compared with its vehicle. Compared with the skin irritation induced by SLS, under these test conditions, calcipotriol is a far weaker irritant on normal human skin. In addition, calcipotriol accelerates stratum corneum turnover to a significantly greater extent than its vehicle.