Saccade Metrics Research Articles

Eye movements reveal sexually dimorphic deficits in children with fetal alcohol spectrum disorder.

Background: We examined the accuracy and characteristics of saccadic eye movements in children with fetal alcohol spectrum disorder (FASD) compared with typically developing control children. Previous studies have found that children with FASD produce saccades that are quantifiably different from controls. Additionally, animal studies have found sex-based differences for behavioral effects after prenatal alcohol exposure. Therefore, we hypothesized that eye movement measures will show sexually dimorphic results.Methods: Children (aged 5–18 years) with FASD (n = 71) and typically developing controls (n = 113) performed a visually-guided saccade task. Saccade metrics and behavior were analyzed for sex and group differences.Results: Female control participants had greater amplitude saccades than control males or females with FASD. Accuracy was significantly poorer in the FASD group, especially in males, which introduced significantly greater variability in the data. Therefore, we conducted additional analyses including only those trials in which the first saccade successfully reached the target within a ± 1° window. In this restricted amplitude dataset, the females with FASD made saccades with significantly lower velocity and longer duration, whereas the males with FASD did not differ from the control group. Additionally, the mean and peak deceleration were selectively decreased in the females with FASD.Conclusions: These data support the hypothesis that children with FASD exhibit specific deficits in eye movement control and sensory-motor integration associated with cerebellar and/or brain stem circuits. Moreover, prenatal alcohol exposure may have a sexually dimorphic impact on eye movement metrics, with males and females exhibiting differential patterns of deficit.

Saccadic eye movement abnormalities in autism spectrum disorder indicate dysfunctions in cerebellum and brainstem.

BackgroundIndividuals with autism spectrum disorder (ASD) show atypical scan paths during social interaction and when viewing faces, and recent evidence suggests that they also show abnormal saccadic eye movement dynamics and accuracy when viewing less complex and non-social stimuli. Eye movements are a uniquely promising target for studies of ASD as their spatial and temporal characteristics can be measured precisely and the brain circuits supporting them are well-defined. Control of saccade metrics is supported by discrete circuits within the cerebellum and brainstem - two brain regions implicated in magnetic resonance (MR) morphometry and histopathological studies of ASD. The functional integrity of these distinct brain systems can be examined by evaluating different parameters of visually-guided saccades.MethodsA total of 65 participants with ASD and 43 healthy controls, matched on age (between 6 and 44-years-old), gender and nonverbal IQ made saccades to peripheral targets. To examine the influence of attentional processes, blocked gap and overlap trials were presented. We examined saccade latency, accuracy and dynamics, as well as the trial-to-trial variability of participants’ performance.ResultsSaccades of individuals with ASD were characterized by reduced accuracy, elevated variability in accuracy across trials, and reduced peak velocity and prolonged duration. In addition, their saccades took longer to accelerate to peak velocity, with no alteration in the duration of saccade deceleration. Gap/overlap effects on saccade latencies were similar across groups, suggesting that visual orienting and attention systems are relatively spared in ASD. Age-related changes did not differ across groups.ConclusionsDeficits precisely and consistently directing eye movements suggest impairment in the error-reducing function of the cerebellum in ASD. Atypical increases in the duration of movement acceleration combined with lower peak saccade velocities implicate pontine nuclei, specifically suggesting reduced excitatory activity in burst cells that drive saccades relative to inhibitory activity in omnipause cells that maintain stable fixation. Thus, our findings suggest that both cerebellar and brainstem abnormalities contribute to altered sensorimotor control in ASD.Electronic supplementary materialThe online version of this article (doi:10.1186/2040-2392-5-47) contains supplementary material, which is available to authorized users.

J26 Portable Oculometric System for Quantitative Assessmentof Horizontal and Vertical Saccades in HD Monitoring

<h3>Background</h3> Saccadic eye movement parameters, such as its dynamics, latency and error rate are considered as a quantitative biomarkers of disease severity in HD. They possess potential for quick, non-invasive, objective assessment of cortical and sub-cortical function and when repeated systematically may allow easy tracking of disease progression or evaluation of novel therapies. Subjective evaluation of saccadic function is already included in UHDRS scale, however, most subtle features of saccadic movement cannot be observed without the instrumentation. On the other hand, application of the oculometric equipment is limited by its significantly high costs and complicated testing procedures. <h3>Methods/techniques</h3> Authors present Saccadometer Research XY, an integrated system dedicated for rapid, quantitative assessment of horizontal and vertical saccadic eye movement metrics in clinical environment. Subject’s eye movements are captured by a small factor, photo-electric sensor, designed for minimal setup and robust measurement. The stimuli is presented by an integrated, 8-targets laser stimuli generator, bound with the subject’s head, so that external stimuli display and head restraint are not required. The system provides pre programmed set of experiments for both reflex and volitional saccadic responses as well as build-in expert system for automatic saccade detection and classification. It allows to complete the testing procedure within several minutes. <h3>Conclusions</h3> Previous Saccadometer models have already proven its usefulness in examination of HD patients. The newest addition allows the measurement of vertical saccades, which are reported now to be more affected in HD than the horizontal.

Effects of mild to moderate sedation on saccadic eye movements

Sedatives alter the metrics of saccadic eye movements. If these effects are nonspecific consequences of sedation, like drowsiness and loss of attention to the task, or differ between sedatives is still unresolved. A placebo-controlled multi-step infusion of one of three sedatives, propofol or midazolam, both GABA-A agonists, or dexmedetedomidine, an α2-adrenergic agonist, was adopted to compare the effects of these three drugs in exactly the same experimental conditions. 60 healthy human volunteers, randomly divided in 4 groups, participated in the study. Each infusion step, delivered by a computer-controlled infusion pump, lasted 20min. During the last 10min of each step, the subject executed a saccadic task. Target concentration was doubled at each step. This block was repeated until the subject was too sedated to continue or for a maximum of 6 blocks. Subjects were unaware which infusion they were receiving. A video eye tracker was used to record the movements of the right eye. Saccadic parameters were modeled as a function of block number, estimated sedative plasma concentration, and subjective evaluation of sedation. Propofol and midazolam had strong effects on the dynamics and latency of the saccades. Midazolam, and to a less extent, propofol, caused saccades to become increasingly hypometric. Dexmedetedomidine had less impact on saccadic metrics and presented no changes in saccadic gain. Suppression of the sympathetic system associated with dexmedetomidine has different effects on eye movements from the increased activity of the inhibitory GABA-A receptors by propofol and midazolam even when the subjects reported similar sedation level.

On the reduced influence of contour on saccade metrics and its competition with stimulus size.

It is well known that the metrical properties of saccadic eye movements are strongly influenced by the extraction of low-level visual features (e.g., luminance). Higher-level visual features (e.g., contour) also play a role, but their relative contribution and time course remain undetermined. Here, we investigated this issue, by testing the influence of contour on saccade metrics. We used a saccade-targeting task in which a peripheral target was, on some trials, simultaneously displayed with a less eccentric distractor. This paradigm is known to yield a global effect, that is a deviation of the eyes towards an intermediate location between the stimuli. The novelty was to test whether this effect would vary with the alignment of the distractor's elementary features. Distractors were of high vs. low luminance, and composed of 16 pixels that were either aligned or misaligned by 0.23° or 0.43°. Our prediction, under the hypothesis that contour intervenes, was that aligned distractors, which formed a definite contour, would deviate the eyes more strongly than misaligned distractors. On the contrary, we found that distractors of high luminance produced greater eye deviations when they were misaligned, and hence more largely spread, than when they were aligned. Furthermore, low-luminance distractors deviated the eyes to the same extent irrespective of their alignment, though showing a reversed, contour-like, effect of alignment for early-triggered saccades. We proposed that contour has only limited influence on saccade metrics, when other, lower-level and more salient visual features, such as the extent of the stimulus pattern, are available.

Saccadic Eye Movement Metrics Reflect Surgical Residents' Fatigue

Little is known about the effects of surgical residents' fatigue on patient safety. We monitored surgical residents' fatigue levels during their call day using (1) eye movement metrics, (2) objective measures of laparoscopic surgical performance, and (3) subjective reports based on standardized questionnaires. Prior attempts to investigate the effects of fatigue on surgical performance have suffered from methodological limitations, including inconsistent definitions and lack of objective measures of fatigue, and nonstandardized measures of surgical performance. Recent research has shown that fatigue can affect the characteristics of saccadic (fast ballistic) eye movements in nonsurgical scenarios. Here we asked whether fatigue induced by time-on-duty (~24 hours) might affect saccadic metrics in surgical residents. Because saccadic velocity is not under voluntary control, a fatigue index based on saccadic velocity has the potential to provide an accurate and unbiased measure of the resident's fatigue level. We measured the eye movements of members of the general surgery resident team at St. Joseph's Hospital and Medical Center (Phoenix, AZ) (6 males and 6 females), using a head-mounted video eye tracker (similar configuration to a surgical headlight), during the performance of 3 tasks: 2 simulated laparoscopic surgery tasks (peg transfer and precision cutting) and a guided saccade task, before and after their call day. Residents rated their perceived fatigue level every 3 hours throughout their 24-hour shift, using a standardized scale. Time-on-duty decreased saccadic velocity and increased subjective fatigue but did not affect laparoscopic performance. These results support the hypothesis that saccadic indices reflect graded changes in fatigue. They also indicate that fatigue due to prolonged time-on-duty does not result necessarily in medical error, highlighting the complicated relationship among continuity of care, patient safety, and fatigued providers. Our data show, for the first time, that saccadic velocity is a reliable indicator of the subjective fatigue of health care professionals during prolonged time-on-duty. These findings have potential impacts for the development of neuroergonomic tools to detect fatigue among health professionals and in the specifications of future guidelines regarding residents' duty hours.

Active Vision in Marmosets: A Model System for Visual Neuroscience

The common marmoset (Callithrix jacchus), a small-bodied New World primate, offers several advantages to complement vision research in larger primates. Studies in the anesthetized marmoset have detailed the anatomy and physiology of their visual system (Rosa et al., 2009) while studies of auditory and vocal processing have established their utility for awake and behaving neurophysiological investigations (Lu et al., 2001a,b; Eliades and Wang, 2008a,b; Osmanski and Wang, 2011; Remington et al., 2012). However, a critical unknown is whether marmosets can perform visual tasks under head restraint. This has been essential for studies in macaques, enabling both accurate eye tracking and head stabilization for neurophysiology. In one set of experiments we compared the free viewing behavior of head-fixed marmosets to that of macaques, and found that their saccadic behavior is comparable across a number of saccade metrics and that saccades target similar regions of interest including faces. In a second set of experiments we applied behavioral conditioning techniques to determine whether the marmoset could control fixation for liquid reward. Two marmosets could fixate a central point and ignore peripheral flashing stimuli, as needed for receptive field mapping. Both marmosets also performed an orientation discrimination task, exhibiting a saturating psychometric function with reliable performance and shorter reaction times for easier discriminations. These data suggest that the marmoset is a viable model for studies of active vision and its underlying neural mechanisms.

Stimulation of the substantia nigra influences the specification of memory-guided saccades

In the absence of sensory information, we rely on past experience or memories to guide our actions. Because previous experimental and clinical reports implicate basal ganglia nuclei in the generation of movement in the absence of sensory stimuli, we ask here whether one output nucleus of the basal ganglia, the substantia nigra pars reticulata (nigra), influences the specification of an eye movement in the absence of sensory information to guide the movement. We manipulated the level of activity of neurons in the nigra by introducing electrical stimulation to the nigra at different time intervals while monkeys made saccades to different locations in two conditions: one in which the target location remained visible and a second in which the target location appeared only briefly, requiring information stored in memory to specify the movement. Electrical manipulation of the nigra occurring during the delay period of the task, when information about the target was maintained in memory, altered the direction and the occurrence of subsequent saccades. Stimulation during other intervals of the memory task or during the delay period of the visually guided saccade task had less effect on eye movements. On stimulated trials, and only when the visual stimulus was absent, monkeys occasionally (∼20% of the time) failed to make saccades. When monkeys made saccades in the absence of a visual stimulus, stimulation of the nigra resulted in a rotation of the endpoints ipsilaterally (∼2°) and increased the reaction time of contralaterally directed saccades. When the visual stimulus was present, stimulation of the nigra resulted in no significant rotation and decreased the reaction time of contralaterally directed saccades slightly. Based on these measurements, stimulation during the delay period of the memory-guided saccade task influenced the metrics of saccades much more than did stimulation during the same period of the visually guided saccade task. Because these effects occurred with manipulation of nigral activity well before the initiation of saccades and in trials in which the visual stimulus was absent, we conclude that information from the basal ganglia influences the specification of an action as it is evolving primarily during performance of memory-guided saccades. When visual information is available to guide the specification of the saccade, as occurs during visually guided saccades, basal ganglia information is less influential.

On the limited effect of stimulus boundaries on saccade metrics

How the endpoint of saccadic eye movements is determined out of many potential peripheral locations is a crucial issue in the field of vision. Models of saccade generation account for this seemingly selective process in terms of competitive interactions between populations of neurons that encode respectively for different saccade amplitudes and directions. However, these models do not specify which visual stimulus properties other than the relative location of the stimuli are involved and how these properties contribute to ultimately determine a single saccade endpoint. We addressed this issue by contrasting the respective contributions of the 2-D spatial extent of the stimuli and the location of their boundaries in a global-effect paradigm. Participants were presented a to-be-looked-at peripheral target stimulus with or without a less eccentric visually invariant distractor. The extent of the target stimulus was manipulated in either one or two dimensions, such that targets differed either by their 2-D spatial extent (small, medium, or large circle) or the location of their boundaries (circle vs. horizontal or vertical ellipse of medium size). Results showed that the distractor deviated the eyes away from the target with the deviation varying with the 2-D spatial extent of the target but not the location of its boundaries. This finding suggests that the spatial distribution of luminance contrast and/or the number of elementary features that compose the stimuli prevails over visual boundaries in specifying the saccade endpoint. Implications for models of saccade generation are discussed.

Does orientation influence saccade metrics irrespective of its relevance for the task?

Does orientation influence saccade metrics irrespective of its relevance for the task?

Altered Accuracy of Saccadic Eye Movements in Children with Fetal Alcohol Spectrum Disorder

Prenatal exposure to alcohol is a major, preventable cause of neurobehavioral dysfunction in children worldwide. The measurement and quantification of saccadic eye movements is a powerful tool for assessing sensory, motor, and cognitive function. The quality of the motor process of an eye movement is known as saccade metrics. Saccade accuracy is 1 component of metrics, which to function optimally requires several cortical brain structures as well as an intact cerebellum and brain-stem. The cerebellum has frequently been reported to be damaged by prenatal alcohol exposure. This study, therefore, tested the hypothesis that children with fetal alcohol spectrum disorder (FASD) will exhibit deficits in the accuracy of saccades. A group of children with FASD (n = 27) between the ages of 8 and 16 and typically developing control children (n = 27) matched for age and sex, completed 3 saccadic eye movement tasks of increasing difficulty. Eye movement performance during the tasks was captured using an infrared eye tracker. Saccade metrics (e.g., velocity, amplitude, accuracy) were quantified and compared between the 2 groups for the 3 different tasks. Children with FASD were more variable in saccade endpoint accuracy, which was reflected by statistically significant increases in the error of the initial saccade endpoint and the frequency of additional, corrective saccades required to achieve final fixation. This increased variability in accuracy was amplified when the cognitive demand of the tasks increased. Children with FASD also displayed a statistically significant increase in response inhibition errors. These data suggest that children with FASD may have deficits in eye movement control and sensory-motor integration including cerebellar circuits, thereby impairing saccade accuracy.

Stimulus properties and saccade metrics: When local features are more critical than global features.

One major issue in the field of vision is how stimulus properties contribute to determine where the eyes move. Here, we examined the role of local versus global visual features in the computation of saccade metrics in the light of the well-known tendency for saccade to vary with the size of the stimuli. We used a saccade-target task in which we varied the properties of a visual distractor simultaneously displayed with the target stimulus. Both the size and the luminance contrast of the distractor were varied but the number of elementary features that composed the distractor was held constant. Our results showed that under such controls mean saccades' landing position remained unaffected by stimulus size irrespective of the level of luminance contrast. These findings suggest that the local visual features of a stimulus may be more critical than global features to specify a particular location to look at. These results are consistent with the notion that local features contribute to determine the amplitude and the width of the neuronal activity patterns associated with the visual stimulation and hence the computation of saccade metrics.

The time course of implicit affective picture processing: An eye movement study.

Consistent with a negativity bias account, neuroscientific and behavioral evidence demonstrates modulation of even early sensory processes by unpleasant, potentially threat-relevant information. The aim of this research is to assess the extent to which pleasant and unpleasant visual stimuli presented extrafoveally capture attention and impact eye movement control. We report an experiment examining deviations in saccade metrics in the presence of emotional image distractors that are close to a nonemotional target. We additionally manipulate the saccade latency to test when the emotional distractor has its biggest impact on oculomotor control. The results demonstrate that saccade landing position was pulled toward unpleasant distractors, and that this pull was due to the quick saccade responses. Overall, these findings support a negativity bias account of early attentional control and call for the need to consider the time course of motivated attention when affect is implicit.

Preparatory neural networks are impaired in adults with attention-deficit/hyperactivity disorder during the antisaccade task.

Adults with attention-deficit/hyperactivity disorder (ADHD) often display executive function impairments, particularly in inhibitory control. The antisaccade task, which measures inhibitory control, requires one to suppress an automatic prosaccade toward a salient visual stimulus and voluntarily make an antisaccade in the opposite direction. ADHD patients not only have longer saccadic reaction times, but also make more direction errors (i.e., a prosaccade was executed toward the stimulus) during antisaccade trials. These deficits may stem from pathology in several brain areas that are important for executive control. Using functional MRI with a rapid event-related design, adults with combined subtype of ADHD (coexistence of attention and hyperactivity problems), who abstained from taking stimulant medication 20 h prior to experiment onset, and age-match controls performed pro- and antisaccade trials that were interleaved with pro- and anti-catch trials (i.e., instruction was presented but no target appeared, requiring no response). This method allowed us to examine brain activation patterns when participants either prepared (during instruction) or executed (after target appearance) correct pro or antisaccades. Behaviorally, ADHD adults displayed several antisaccade deficits, including longer and more variable reaction times and more direction errors, but saccade metrics (i.e., duration, velocity, and amplitude) were normal. When preparing to execute an antisaccade, ADHD adults showed less activation in frontal, supplementary, and parietal eye fields, compared to controls. However, activation in these areas was normal in the ADHD group during the execution of a correct antisaccade. Interestingly, unlike controls, adults with ADHD produced greater activation than controls in dorsolateral prefrontal cortex during antisaccade execution, perhaps as part of compensatory mechanisms to optimize antisaccade production. Overall, these data suggest that the saccade deficits observed in adults with ADHD do not result from an inability to execute a correct antisaccade but rather the failure to properly prepare (i.e., form the appropriate task set) for the antisaccade trial. The data support the view that the executive impairments, including inhibitory control, in ADHD adults are related to poor response preparation.

Peripheral spatial frequency processing affects timing and metrics of saccades

Peripheral spatial frequency processing affects timing and metrics of saccades

I04 Palm-size oculomotor laboratory for field testing of saccadic system potentially applicable for monitoring of HD development

Saccadic eye movement measurement is reported as potential sensitive biomarker of Huntington's disease (HD), in both pre-manifest and manifest stages. While definitely less specific than imagining techniques, it possess potential for quick, low cost and non invasive quantitative assessment of brain functional status. It is also less prone to neuronal compensation mechanism which continues to conceal the evidence of functional motor impairment, especially when voluntary component is involved in saccadic test paradigm (antisaccade-peripherial-conflict). According to recent findings, the changes of saccadic metrics accompany HD progression and they can be considered as a means of tracking the current patient condition and outcome of new therapeutic interventions. Authors present portable, integrated diagnostic environment, Saccadometer Clinical. The system is designed as minimal-setup for evaluation of saccadic metrics, allowing to establish subjects functional deterioration profile based on the repetitive measurements (status samples) accumulated over the time. The key component is small factor, battery powered, head-mounted intelligent sensor, capable of generating laser dot stimuli for both reflex and volitional decision based saccadic tests, and performing automated eye movement acquisition and classification. Due to portability, ease of use, and build-in telemetry link for wireless communication, the measurement can be carried by patient her/himself in home environment, providing facility for continuous, remote monitoring of patient's functional status. In order to broaden the spectrum of measured parameters, current development efforts aim to include the smooth pursuit and the vestibule-ocular (postural sway) tests within the same miniaturised system, serving as complementary biomarkers of HD.

Linking visual response properties in the superior colliculus to saccade behavior

Here we examined the influence of the visual response in the superior colliculus (SC) (an oculomotor control structure integrating sensory, motor and cognitive signals) on the development of the motor command that drives saccadic eye movements in monkeys. We varied stimulus luminance to alter the timing and magnitude of visual responses in the SC and examined how these changes correlated with resulting saccade behavior. Increasing target luminance resulted in multiple modulations of the visual response, including increased magnitude and decreased response onset latency. These signal modulations correlated strongly with changes in saccade latency and metrics, indicating that these signal properties carry through to the neural computations that determine when, where and how fast the eyes will move. Thus, components of the earliest part of the visual response in the SC provide important building blocks for the neural basis of the sensory-motor transformation, highlighting a critical link between the properties of the visual response and saccade behavior.

When larger visual distractors become less disruptive: Behavioral evidence for lateral inhibition in saccade generation

How neuronal activity is integrated over time may largely rely on excitatory and inhibitory mechanisms. Dynamic neural field models assume that local excitation and lateral inhibition (i.e., the "Mexican hat") shape the output of neural networks. Most models of saccade generation assume that such interactions in the superior colliculus play a key role in determining both the metrics and the latency of saccades. Here, we investigated the role of lateral inhibition in saccade metrics in humans. We used a saccade target task in which a visual distractor line was presented close to a peripheral visual target (i.e., a small circle). Models assuming lateral inhibition predict that beyond a critical size larger distractors induce less perturbation than smaller ones. To assess this prediction, we varied the length of the distractor. Results confirmed that a distractor presented along with the target deviated the saccade's landing position away from the target. This perturbation increased with distractor length but only up to a critical size as the effect reversed for larger distractors, leading to a reduced perturbation on saccade metrics. These results suggest that larger distractors induce a neuronal activity pattern wide enough to involve lateral inhibition, thereby decreasing the distractor's weight in the spatial integration of distractor and target locations. They are consistent with a critical role of lateral inhibition in the computation of saccade metrics.

Remapping of the line motion illusion across eye movements

Although motion processing in the brain has been classically studied in terms of retinotopically defined receptive fields, recent evidence suggests that motion perception can occur in a spatiotopic reference frame. We investigated the underlying mechanisms of spatiotopic motion perception by examining the role of saccade metrics as well as the capacity of trans-saccadic motion. To this end, we used the line motion illusion (LMI), in which a straight line briefly shown after a high contrast stimulus (inducer) is perceived as expanding away from the inducer position. This illusion provides an interesting test of spatiotopic motion because the neural correlates of this phenomenon have been found early in the visual cortex and the effect does not require focused attention. We measured the strength of LMI both with stable fixation and when participants were asked to perform a 10° saccade during the blank ISI between the inducer and the line. A strong motion illusion was found across saccades in spatiotopic coordinates. When the inducer was presented near in time to the saccade cue, saccadic latencies were longer, saccade amplitudes were shorter, and the strength of reported LMI was consistently reduced. We also measured the capacity of the trans-saccadic LMI by varying the number of inducers. In contrast to a visual-spatial memory task, we found that the LMI was largely eliminated by saccades when two or more inducers were displayed. Together, these results suggest that motion perceived in non-retinotopic coordinates depends on an active, saccade-dependent remapping process with a strictly limited capacity.

A closer look at visually guided saccades in autism and Asperger’s disorder

Motor impairments have been found to be a significant clinical feature associated with autism and Asperger’s disorder (AD) in addition to core symptoms of communication and social cognition deficits. Motor deficits in high-functioning autism (HFA) and AD may differentiate these disorders, particularly with respect to the role of the cerebellum in motor functioning. Current neuroimaging and behavioral evidence suggests greater disruption of the cerebellum in HFA than AD. Investigations of ocular motor functioning have previously been used in clinical populations to assess the integrity of the cerebellar networks, through examination of saccade accuracy and the integrity of saccade dynamics. Previous investigations of visually guided saccades in HFA and AD have only assessed basic saccade metrics, such as latency, amplitude, and gain, as well as peak velocity. We used a simple visually guided saccade paradigm to further characterize the profile of visually guided saccade metrics and dynamics in HFA and AD. It was found that children with HFA, but not AD, were more inaccurate across both small (5°) and large (10°) target amplitudes, and final eye position was hypometric at 10°. These findings suggest greater functional disturbance of the cerebellum in HFA than AD, and suggest fundamental difficulties with visual error monitoring in HFA.