Glial immune-related pathways mediate effects of closed head traumatic brain injury on behavior and lethality in Drosophila.

In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, we developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, we examined TBI-dependent behavior and lethality in mutants of the master immune regulator NF-κB, important for AMP induction, and found that while sleep and motor function effects were reduced, lethality effects were enhanced. Similarly, loss of most AMP classes also renders flies susceptible to lethal TBI effects. These studies validate a new Drosophila TBI model and identify immune pathways as in vivo mediators of TBI effects.

A deep sleep stage in Drosophila with a functional role in waste clearance.

Sleep is a highly conserved state, suggesting that sleep's benefits outweigh the increased vulnerability it brings. Yet, little is known about how sleep fulfills its functions. Here, we used video tracking in tethered flies to identify a discrete deep sleep stage in Drosophila, termed proboscis extension sleep, that is defined by repeated stereotyped proboscis extensions and retractions. Proboscis extension sleep is accompanied by highly elevated arousal thresholds and decreased brain activity, indicative of a deep sleep state. Preventing proboscis extensions increases injury-related mortality and reduces waste clearance. Sleep deprivation reduces waste clearance and during subsequent rebound sleep, sleep, proboscis extensions, and waste clearance are increased. Together, these results provide evidence of a discrete deep sleep stage that is linked to a specific function and suggest that waste clearance is a core and ancient function of deep sleep.

Oscillatory brain activity in spontaneous and induced sleep stages in flies.

Sleep is a dynamic process comprising multiple stages, each associated with distinct electrophysiological properties and potentially serving different functions. While these phenomena are well described in vertebrates, it is unclear if invertebrates have distinct sleep stages. We perform local field potential (LFP) recordings on flies spontaneously sleeping, and compare their brain activity to flies induced to sleep using either genetic activation of sleep-promoting circuitry or the GABAA agonist Gaboxadol. We find a transitional sleep stage associated with a 7-10 Hz oscillation in the central brain during spontaneous sleep. Oscillatory activity is also evident when we acutely activate sleep-promoting neurons in the dorsal fan-shaped body (dFB) of Drosophila. In contrast, sleep following Gaboxadol exposure is characterized by low-amplitude LFPs, during which dFB-induced effects are suppressed. Sleep in flies thus appears to involve at least two distinct stages: increased oscillatory activity, particularly during sleep induction, followed by desynchronized or decreased brain activity.

Human gaze following response is affected by visual acuity.

The present study investigated how gaze following eye movements are affected by stimulus contrast and spatial frequency and by aberrations in central visual acuity due to refractive errors. We measured 30 healthy subjects with a range of visual acuities but without any refractive correction. Visual acuity was tested using a Landolt-C chart. Subjects were divided into three groups with low, intermediate, or good visual acuity. Gaze following responses (GFR) to moving Gabor patches were recorded by video-oculography. In each trial, the subjects were presented with a single Gabor patch with a specific spatial frequency and luminance contrast that moved sinusoidally in the horizontal plane. We observed that GFR gain decreased with increasing spatial frequency and decreasing contrast and was correlated with visual acuity. GFR gain was lower and decreased more for subjects with lower visual acuity; this was especially so for lower stimulus contrasts that are not tested in standard acuity tests. The largest differences between the groups were observed at spatial frequencies around 4 cpd and at contrasts up to 10%. Aberrations in central visual acuity due to refractive errors affect the GFR response depending on the contrast and spatial frequency of the moving stimulus. Measuring this effect may contribute to a better estimate of changes in visual function as a result of aging, disease, or treatments meant to improve vision.

Knock, knock to reset the clock: mechanosensation and circadian rhythms.

Circadian clocks, which underlie the daily rhythms in virtually all organisms, are entrained by diurnal changes in light, temperature, nutrients, and even sound. Simoni et al. (2014) demonstrate that diurnal variation in mechanical vibrations can reset circadian clock phase, providing a potential mechanism for integrating diverse clock-entraining stimuli.

A dynamic deep sleep stage in Drosophila.

How might one determine whether simple animals such as flies sleep in stages? Sleep in mammals is a dynamic process involving different stages of sleep intensity, and these are typically associated with measurable changes in brain activity (Blake and Gerard, 1937; Rechtschaffen and Kales, 1968; Webb and Agnew, 1971). Evidence for different sleep stages in invertebrates remains elusive, even though it has been well established that many invertebrate species require sleep (Campbell and Tobler, 1984; Hendricks et al., 2000; Shaw et al., 2000; Sauer et al., 2003). Here we used electrophysiology and arousal-testing paradigms to show that the fruit fly, Drosophila melanogaster, transitions between deeper and lighter sleep within extended bouts of inactivity, with deeper sleep intensities after ∼15 and ∼30 min of inactivity. As in mammals, the timing and intensity of these dynamic sleep processes in flies is homeostatically regulated and modulated by behavioral experience. Two molecules linked to synaptic plasticity regulate the intensity of the first deep sleep stage. Optogenetic upregulation of cyclic adenosine monophosphate during the day increases sleep intensity at night, whereas loss of function of a molecule involved in synaptic pruning, the fragile-X mental retardation protein, increases sleep intensity during the day. Our results show that sleep is not homogenous in insects, and suggest that waking behavior and the associated synaptic plasticity mechanisms determine the timing and intensity of deep sleep stages in Drosophila.

A sleep/wake circuit controls isoflurane sensitivity in Drosophila.

General anesthesia remains a mysterious phenomenon, even though a number of compelling target proteins and processes have been proposed [1]. General anesthetics such as isoflurane abolish behavioral responsiveness in all animals, and in the mammalian brain, these diverse compounds probably achieve this in part by targeting endogenous sleep mechanisms [2, 3]. However, most animals sleep [4], and they are therefore likely to have conserved sleep processes. A decade of neurogenetic studies of arousal in Drosophila melanogaster have identified a number of different neurons and brain structures that modulate sleep duration in the fly brain [5-9], but it has remained unclear until recently whether any neurons might form part of a dedicated circuit that actively controls sleep and wake states in the fly brain, as has been proposed for the mammalian brain [10]. We studied general anesthesia in Drosophila by measuring stimulus-induced locomotion under isoflurane gas exposure. Using a syntaxin1A gain-of-function construct, we found that increasing synaptic activity in different Drosophila neurons could produce hypersensitivity or resistance to isoflurane. We uncover a common pathway in the fly brain controlling both sleep duration and isoflurane sensitivity, centered on monoaminergic modulation of sleep-promoting neurons of the fan-shaped body.

Drosophila strategies to study psychiatric disorders.

For decades, Drosophila melanogaster has been used as a model organism to study human diseases, ranging from heart disease to cancer to neurological disorders [9]. For studying neurodegenerative diseases, Drosophila has been instrumental in understanding disease mechanisms and pathways as well as being an efficient tool in drug discovery studies. For some better-understood disorders, such as Fragile X (a mental retardation syndrome), clinical trials are being run, based in part on translational work in flies and rodents. However, Drosophila is currently less used to study psychiatric disorders such as autism, schizophrenia and attention deficit and hyperactivity disorder (ADHD), despite numerous discoveries of disease susceptibility genes that could be explored by reverse genetics or miss-expression studies. This deficit might be explained by (1) a lack of reliable tests to study more complex disease (endo)phenotypes in flies, (2) difficulties in translating disease symptoms into animal models and (3) the polygenetic nature of these diseases. In this review we discuss strategies to use D. melanogaster to study complex psychiatric disorders such as schizophrenia, autism and ADHD. Two common features of these diseases may be defective sleep and attention mechanisms, hence calling for better methods for quantifying and screening arousal thresholds in flies.

Accelerated loss of hearing and vision in the DNA-repair deficient Ercc1(δ/-) mouse.

Age-related loss of hearing and vision are two very common disabling conditions, but the underlying mechanisms are still poorly understood. Damage by reactive oxygen species and other reactive cellular metabolites, which in turn may damage macromolecules such as DNA, has been implicated in both processes. To investigate whether DNA damage can contribute to age-related hearing and vision loss, we investigated hearing and vision in Ercc1(δ/-) mutant mice, which are deficient in DNA repair of helix-distorting DNA lesions and interstrand DNA crosslinks. Ercc1(δ/-) mice showed a progressive, accelerated increase of hearing level thresholds over time, most likely arising from deteriorating cochlear function. Ercc1(δ/-) mutants also displayed a progressive decrease in contrast sensitivity followed by thinning of the outer nuclear layer of the eyeball. The strong parallels with normal ageing suggest that unrepaired DNA damage can induce age-related decline of the auditory and visual system.

Three-dimensional optokinetic eye movements in the C57BL/6J mouse.

PURPOSE: To study three-dimensional optokinetic eye movements of wild-type C57BL/6J mice, the most commonly used mouse in oculomotor physiology. Optokinetic eye movements are reflexive eye movements that use visual feedback to minimize image motion across the retina. These gaze-stabilizing reflexes are a prominent model system for studying motor control and learning. They are three dimensional and consist of a horizontal, vertical, and torsional component. METHODS: Eye movements were evoked by sinusoidally rotating a virtual sphere of equally spaced dots at six frequencies (0.1-1 Hz), with a fixed amplitude of 5 degrees . Markers were applied to the mouse eye and video oculography was used to record its movements in three dimensions. In addition, marker tracking was compared with conventional pupil tracking of horizontal optokinetic eye movements. RESULTS: Gains recorded with marker and pupil tracking are not significantly different. Optokinetic eye movements in mice are equally well developed in all directions and have a uniform input-output relation for all stimuli, including stimuli that evoke purely torsional eye movements, with gains close to unity and minimal phase differences. CONCLUSIONS: Optokinetic eye movements of C57Bl6 mice largely compensate for image motion over the retina, regardless of stimulus orientation. All responses are frequency-velocity dependent: gains decrease and phase lags increase with increasing stimulus frequency. Mice show strong torsional responses, with high gains at low stimulus frequency.

Age- and sex-related differences in contrast sensitivity in C57BL/6 mice.

PURPOSE: To measure contrast sensitivity in C57BL/6, the most commonly used mouse in behavioral neuroscience, and to study the effect of sex, age, and miotic drugs on the contrast sensitivity function. In addition, the authors tested a mutant in which plasticity in the cerebellum is impaired by expressing a protein kinase C inhibitor. This inhibitor is also expressed in the retina, possibly affecting vision. METHODS: The gain of the optokinetic reflex (OKR) decreases as stimuli become more difficult to see. Recording OKR gains evoked by moving sine gratings shows whether the stimulus was distinguished from a homogeneous background and how well the stimulus was distinguished. RESULTS: Female mice have lower OKR gains than male mice (both groups: n = 10, P = 0.001). A similar difference was observed between 4-month-old (n = 10) and 9-month-old (n = 5) C57Bl/6 mice (P = 0.001). These differences could not be detected with earlier dichotomic tests. C57BL/6 mice are able to see contrasts as low as 1%, well below the previously reported 5% threshold. Pilocarpine had no significant effect on contrast sensitivity (both groups: n = 10, P = 0.89). Vision in L7-PKCi mutants was unaffected (both groups: n = 10, P = 0.82). CONCLUSIONS: OKR gains decrease as stimuli become more difficult to see, making the OKR a powerful tool to quantify contrast sensitivity. In C57BL/6 these response magnitudes vary greatly between sexes and between mice that differ only a few months in age. Therefore, it is important to match groups according to age and sex in experiments that require unimpaired vision. Otherwise, impaired vision can be misinterpreted as a learning or motor problem.