Regional specificity of MRI contrast parameter changes in normal ageing revealed by voxel-based quantification (VBQ).

Normal ageing is associated with characteristic changes in brain microstructure. Although in vivo neuroimaging captures spatial and temporal patterns of age-related changes of anatomy at the macroscopic scale, our knowledge of the underlying (patho)physiological processes at cellular and molecular levels is still limited. The aim of this study is to explore brain tissue properties in normal ageing using quantitative magnetic resonance imaging (MRI) alongside conventional morphological assessment. Using a whole-brain approach in a cohort of 26 adults, aged 18-85years, we performed voxel-based morphometric (VBM) analysis and voxel-based quantification (VBQ) of diffusion tensor, magnetization transfer (MT), R1, and R2* relaxation parameters. We found age-related reductions in cortical and subcortical grey matter volume paralleled by changes in fractional anisotropy (FA), mean diffusivity (MD), MT and R2*. The latter were regionally specific depending on their differential sensitivity to microscopic tissue properties. VBQ of white matter revealed distinct anatomical patterns of age-related change in microstructure. Widespread and profound reduction in MT contrasted with local FA decreases paralleled by MD increases. R1 reductions and R2* increases were observed to a smaller extent in overlapping occipito-parietal white matter regions. We interpret our findings, based on current biophysical models, as a fingerprint of age-dependent brain atrophy and underlying microstructural changes in myelin, iron deposits and water. The VBQ approach we present allows for systematic unbiased exploration of the interaction between imaging parameters and extends current methods for detection of neurodegenerative processes in the brain. The demonstrated parameter-specific distribution patterns offer insights into age-related brain structure changes in vivo and provide essential baseline data for studying disease against a background of healthy ageing.

Magnetic resonance imaging of Huntington's disease: preparing for clinical trials.

The known genetic mutation causing Huntington's disease (HD) makes this disease an important model to study links between gene and brain function. An autosomal dominant family history and the availability of a sensitive and specific genetic test allow pre-clinical diagnosis many years before the onset of any typical clinical signs. This review summarizes recent magnetic resonance imaging (MRI)-based findings in HD with a focus on the requirements if imaging is to be used in treatment trials. Despite its monogenetic cause, HD presents with a range of clinical manifestations, not explained by variation in the number of CAG repeats in the affected population. Neuroimaging studies have revealed a complex pattern of structural and functional changes affecting widespread cortical and subcortical regions far beyond the confines of the striatal degeneration that characterizes this disorder. Besides striatal dysfunction, functional imaging studies have reported a variable pattern of increased and decreased activation in cortical regions in both pre-clinical and clinically manifest HD-gene mutation carriers. Beyond regional brain activation changes, evidence from functional and diffusion-weighted MRI further suggests disrupted connectivity between corticocortical and corticostriatal areas. However, substantial inconsistencies with respect to structural and functional changes have been reported in a number of studies. Possible explanations include methodological factors and differences in study samples. There may also be biological explanations but these are poorly characterized and understood at present. Additional insights into this phenotypic variability derived from study of mouse models are presented to explore this phenomenon.

Genotype-phenotype interactions in primary dystonias revealed by differential changes in brain structure.

Our understanding of how genotype determines phenotype in primary dystonia is limited. Familial young-onset primary dystonia is commonly due to the DYT1 gene mutation. A critical question, given the 30% penetrance of clinical symptoms in DYT1 mutation carriers, is why the same genotype leads to differential clinical expression and whether non-DYT1 adult-onset primary dystonia, with and without family history share pathophysiological mechanisms with DYT1 dystonia. This study examines the relationship between dystonic phenotype and the DYT1 gene mutation by monitoring whole-brain structure using voxel-based morphometry. We acquired magnetic resonance imaging data of symptomatic and asymptomatic DYT1 mutation carriers, of non-DYT1 primary dystonia patients, with and without family history and control subjects with normal DYT1 alleles. By crossing the factors genotype and phenotype we demonstrate a significant interaction in terms of brain anatomy confined to the basal ganglia bilaterally. The explanation for this effect differs according to both gene and dystonia status: non-DYT1 adult-onset dystonia patients and asymptomatic DYT1 carriers have significantly larger basal ganglia compared to healthy subjects and symptomatic DYT1 mutation carriers. There is a significant negative correlation between severity of dystonia and basal ganglia size in DYT1 mutation carriers. We propose that differential pathophysiological and compensatory mechanisms lead to brain structure changes in non-DYT1 primary adult-onset dystonias and DYT1 gene carriers. Given the range of age of onset, there may be differential genetic modulation of brain development that in turn determines clinical expression. Alternatively, a DYT1 gene dependent primary defect of motor circuit development may lead to stress-induced remodelling of the basal ganglia and hence dystonia.

Automatic detection of preclinical neurodegeneration: presymptomatic Huntington disease.

BACKGROUND: Treatment of neurodegenerative diseases is likely to be most beneficial in the very early, possibly preclinical stages of degeneration. We explored the usefulness of fully automatic structural MRI classification methods for detecting subtle degenerative change. The availability of a definitive genetic test for Huntington disease (HD) provides an excellent metric for judging the performance of such methods in gene mutation carriers who are free of symptoms. METHODS: Using the gray matter segment of MRI scans, this study explored the usefulness of a multivariate support vector machine to automatically identify presymptomatic HD gene mutation carriers (PSCs) in the absence of any a priori information. A multicenter data set of 96 PSCs and 95 age- and sex-matched controls was studied. The PSC group was subclassified into three groups based on time from predicted clinical onset, an estimate that is a function of DNA mutation size and age. RESULTS: Subjects with at least a 33% chance of developing unequivocal signs of HD in 5 years were correctly assigned to the PSC group 69% of the time. Accuracy improved to 83% when regions affected by the disease were selected a priori for analysis. Performance was at chance when the probability of developing symptoms in 5 years was less than 10%. CONCLUSIONS: Presymptomatic Huntington disease gene mutation carriers close to estimated diagnostic onset were successfully separated from controls on the basis of single anatomic scans, without additional a priori information. Prior information is required to allow separation when degenerative changes are either subtle or variable.

Putaminal activity is related to perceptual certainty.

We have investigated the neural basis of perceptual certainty using a simple discrimination paradigm. Psychophysical experiments have shown that a pair of identical electrical stimuli to the skin or a pair of auditory clicks to the ears are consistently perceived as two separate events in time when the inter-stimulus interval (ISIs) is long, and perceived as simultaneous events when the ISIs are very short. The perceptual certainty of having received one or two stimuli decreases when the ISI lies between these two extremes and this is reflected in inconsistent reporting of the percept across trials. In two fMRI experiments, 14 healthy subjects received either paired electrical pulses delivered to the forearm (ISIs=5-110 ms) or paired auditory clicks presented binaurally (ISIs=1-20 ms). For each subject and modality, we calculated a consistency index (CI) representing the level of perceptual certainty. The task activated pre-SMA and anterior cingulate cortex, plus the cerebellum and the basal ganglia. Critically, activity in the right putamen was linearly dependent on CI for both tactile and auditory discrimination, with topographically distinct effects in the two modalities. These results support a role for the human putamen in the "automatic" perception of temporal features of tactile and auditory stimuli.

Anti-basal ganglia antibodies and Tourette's syndrome: a voxel-based morphometry and diffusion tensor imaging study in an adult population.

Anti-basal ganglia antibodies (ABGAs) have been suggested to be a hallmark of autoimmunity in Gilles de la Tourette's syndrome (GTS), possibly related to prior exposure to streptococcal infection. In order to detect whether the presence of ABGAs was associated with subtle structural changes in GTS, whole-brain analysis using independent sets of T(1) and diffusion tensor imaging MRI-based methods were performed on 22 adults with GTS with (n = 9) and without (n = 13) detectable ABGAs in the serum. Voxel-based morphometry analysis failed to detect any significant difference in grey matter density between ABGA-positive and ABGA-negative groups in caudate nuclei, putamina, thalami and frontal lobes. These results suggest that ABGA synthesis is not related to structural changes in grey and white matter (detectable with these methods) within frontostriatal circuits.

The neural basis of temporal auditory discrimination.

When two identical stimuli, such as a pair of clicks, are presented with a sufficiently long time-interval between them they are readily perceived as two separate events. However, as they are presented progressively closer together, there comes a point when the two separate stimuli are perceived as one. This phenomenon applies not only to hearing but also to other sensory modalities. Damage to the basal ganglia disturbs this type of temporal discrimination irrespective of sensory modality, suggesting a multimodal process is involved. Our aim was to study the neural substrate of auditory temporal discrimination in healthy subjects and to compare it with structures previously associated with analogous tactile temporal discrimination. During fMRI scanning, paired-clicks separated by variable inter-stimulus intervals (1-50 ms) were delivered binaurally, with different intensities delivered to each ear, yielding a lateralised auditory percept. Subjects were required (a) to report whether they heard one or two stimuli (TD: temporal discrimination); or (b) to report whether the stimuli were located on the right or left side of the head mid-line (SD: spatial discrimination); or (c) simply to detect the presence of an auditory stimulus (control task). Our results showed that both types of auditory discrimination (TD and SD) compared to simple detection activated a network of brain areas including regions of prefrontal cortex and basal ganglia. Critically, two clusters in pre-SMA and the anterior cingulate cortex were specifically activated by TD. Furthermore, these clusters overlap with regions activated for similar judgments in the tactile modality suggesting that they fulfill a multimodal function in the temporal processing of sensory events.

Attention to pain localization and unpleasantness discriminates the functions of the medial and lateral pain systems.

Functional imaging studies have identified a matrix of structures in the brain that respond to noxious stimuli. Within this matrix, a division of function between sensory-discriminative and affective responses has so far been demonstrated by manipulating either pain intensity or unpleasantness under hypnosis in two different normal volunteer groups studied on separate occasions. Our study used positron emission tomography (PET) to demonstrate this division of function under more natural conditions in a healthy group of volunteers, using a CO(2) laser to provide nociceptive stimuli that selectively activate A-delta and C-fibres without contamination by touch sensations. We measured the differential cerebral responses to noxious and innocuous laser stimuli during conditions of selective attention to either the unpleasantness or location of the stimuli. Attention to location increased responses in the contralateral (right) primary somatosensory and inferior parietal cortices. This result implies that these components of the lateral pain system are concerned mainly with the localization of pain. In contrast, attention to unpleasantness increased responses in bilateral perigenual cingulate and orbitofrontal cortices, contralateral (right) amygdala, ipsilateral (left) hypothalamus, posterior insula, M1 and frontal pole. These areas comprise key components of the medial pain and neuroendocrine systems and the results suggest that they have a role in the affective response to pain. Our results indicate the importance of attentional effects on the pattern of nociceptive processing in the brain. They also provide the first clear demonstration, within a single experiment, of a major division of function within the neural pain matrix.

A PET study exploring the laterality of brainstem activation in migraine using glyceryl trinitrate.

Migraine is a common disabling condition likely to be associated with dysfunction of brain pathways involved in pain and other sensory modalities. A cardinal, indeed signature, feature of the disorder that led to its name is that the pain may be lateralized. H(2)15O-labelled PET was used to study 24 migraineurs and eight healthy controls. The migraineurs were divided into three groups according to the site of their headache: right, left or bilateral. In each group, a migraine was induced using a glyceryl trinitrate (GTN) infusion. The subjects were scanned at predefined points: pre-infusion, during GTN, during migraine and post-migraine. SPM99 software was used to analyse the data. Significant brainstem activation was seen in the dorsal lateral pons (P < 0.05 after small volume correction) during the migraine state versus the pain-free state when comparing migraineurs with controls. When each group was analysed separately, to investigate laterality, it was found that the dorsal pontine activation was ipsilateral in the right-sided and left-sided groups and bilateral in the bilateral headache group with a left-sided preponderance. Consistent with previous work, the activation persisted after pain was controlled by sumatriptan. These results suggest that lateralization of pain in migraine is due to lateralized brain dysfunction.

Superior temporal sulcus anatomical abnormalities in childhood autism: a voxel-based morphometry MRI study.

The underlying neurobiology of autism, a severe pervasive developmental disorder, remains unknown. Few neocortical brain MRI abnormalities have been reported. Using rest functional brain imaging, two independent studies have described localized bilateral temporal hypoperfusion in children with primary autism. In order to search for convergent evidence of anatomical abnormalities in autistic children, we performed an anatomical MRI study using optimized whole-brain voxel-based morphometry (VBM). High-resolution 3-D T1-weighted MRI data sets were acquired in 21 children with primary autism (mean age 9.3 +/- 2.2 years) and 12 healthy control children (mean age 10.8 +/- 2.7 years). By comparing autistic children to normal children, we found bilaterally significant decreases of grey matter concentration located in superior temporal sulcus (STS) (P < 0.05 corrected, after small volume correction; SVC). Children with autism were also found to have a decrease of white matter concentration located in the right temporal pole and in cerebellum (P < 0.05, corrected) compared to normal children. These results suggest that autism is associated with bilateral anatomical abnormalities localized in the STS and are remarkably consistent with functional hypoperfusion previously reported in children with autism. The multimodal STS areas are involved in highest level of cortical integration of both sensory and limbic information. Moreover, the STS is now recognized as a key cortical area of the "social brain" and is implicated in social perceptual skills that are characteristically impaired in autism. Therefore, the convergent anatomical and functional temporal abnormalities observed in autism may be important in the understanding of brain behavior relationships in this severe developmental disorder.

Neural correlates of motor recovery after stroke: a longitudinal fMRI study.

Recovery of motor function after stroke may occur over weeks or months and is often attributed to cerebral reorganization. We have investigated the longitudinal relationship between recovery after stroke and task-related brain activation during a motor task as measured using functional MRI (fMRI). Eight first-ever stroke patients presenting with hemiparesis resulting from cerebral infarction sparing the primary motor cortex, and four control subjects were recruited. Subjects were scanned on a number of occasions whilst performing an isometric dynamic visually paced hand grip task. Recovery in the patient group was assessed using a battery of outcome measures at each time point. Task-related brain activations decreased over sessions as a function of recovery in a number of primary and non-primary motor regions in all patients, but no session effects were seen in the controls. Furthermore, consistent decreases across sessions correlating with recovery were seen across the whole patient group independent of rate of recovery or initial severity, in primary motor cortex, premotor and prefrontal cortex, supplementary motor areas, cingulate sulcus, temporal lobe, striate cortex, cerebellum, thalamus and basal ganglia. Although recovery-related increases were seen in different brain regions in four patients, there were no consistent effects across the group. These results further our understanding of the recovery process by demonstrating for the first time a clear temporal relationship between recovery and task-related activation of the motor system after stroke.

Neural correlates of voluntary breathing in humans.

To investigate the functional neuroanatomy of voluntary respiratory control, blood O2 level-dependent functional magnetic resonance imaging was performed in six healthy right-handed individuals during voluntary hyperpnea. Functional images of the whole brain were acquired during 30-s periods of spontaneous breathing alternated with 30-s periods of isocapnic hyperpnea [spontaneous vs. voluntary: tidal volume = 0.5 +/- 0.01 vs. 1.3 +/- 0.1 (SE) liters and breath duration = 4.0 +/- 0.4 vs. 3.2 +/- 0.4 (SE) s]. For the group, voluntary hyperpnea was associated with significant (P < 0.05, corrected for multiple comparisons) neural activity bilaterally in the primary sensory and motor cortices, supplementary motor area, cerebellum, thalamus, caudate nucleus, and globus pallidum. Significant increases in activity were also identified in the medulla (corrected for multiple comparisons on the basis of a small volume correction for a priori region of interest) in a superior dorsal position (P = 0.012). Activity within the medulla suggests that the brain stem respiratory centers may have a role in mediating the voluntary control of breathing in humans.

Neural correlates of outcome after stroke: a cross-sectional fMRI study.

Recovery of motor function after stroke may occur over weeks or months and is often attributed to neuronal reorganization. Functional imaging studies investigating patients who have made a good recovery after stroke have suggested that recruitment of other motor-related networks underlies this recovery. However, patients with less complete recovery have rarely been studied, or else the degree of recovery has not been taken into account. We set out to investigate the relationship between the degree of recovery after stroke and the pattern of recruitment of brain regions during a motor task as measured using functional MRI. We recruited 20 patients who were at least 3 months after their first ever stroke, and 26 right-handed age-matched control subjects. None of our patients had infarcts involving the hand region of the primary motor cortex. All subjects were scanned whilst performing an isometric, dynamic visually paced handgrip task. The degree of functional recovery of each patient was assessed using a battery of outcome measures. Single-patient versus control group analysis revealed that patients with poor recovery were more likely to recruit a number of motor-related brain regions over and above those seen in the control group during the motor task, whereas patients with more complete recovery were more likely to have 'normal' task-related brain activation. Across the whole patient group and across stroke subtypes, we were able to demonstrate a negative correlation between outcome and the degree of task-related activation in regions such as the supplementary motor area, cingulate motor areas, premotor cortex, posterior parietal cortex, and cerebellum. This negative correlation was also seen in parts of both contralateral and ipsilateral primary motor cortex. These results further our understanding of the recovery process by demonstrating for the first time a clear relationship between task-related activation of the motor system and outcome after stroke.

Age-related changes in the neural correlates of motor performance.

Age-related neurodegenerative and neurochemical changes are thought to underlie decline in motor and cognitive functions, but compensatory processes in cortical and subcortical function may allow maintenance of performance level in some people. Our objective was to investigate age-related changes in the motor system of the human brain using functional MRI. Twenty six right handed volunteers were scanned whilst performing an isometric, dynamic, visually paced hand grip task, using dominant (right) and non-dominant (left) hands in separate sessions. Hand grip with visual feedback activated a network of cortical and subcortical regions known to be involved in the generation of simple motor acts. In addition, activation was seen in a putative human 'grasping circuit', involving rostral ventral premotor cortex (Brodmann area 44) and intraparietal sulcus. Within this network, a number of regions were more likely to be activated the older the subject. In particular, age-related changes in task- specific activations were demonstrated in left deep anterior central sulcus when using the dominant or non-dominant hand. Additional age-related increases were seen in caudal dorsal premotor cortex, caudal cingulate sulcus, intraparietal sulcus, insula, frontal operculum and cerebellar vermis. We have demonstrated a clear age-related effect in the neural correlates of motor performance, and furthermore suggest that these changes are non-linear. These results support the notion that an adaptable and plastic motor network is able to respond to age-related degenerative changes in order to maintain performance levels.

Differential brain activations during intentionally simulated and subjectively experienced paralysis.

INTRODUCTION: Distinguishing conversion disorder from malingering presents a significant challenge as the diagnosis ultimately depends on the patient's subjective report and the clinician's suspicion of an intention to deceive. Using hypnosis to manipulate the intentionality of movement inhibition in the same subjects, we used positron emission tomography (PET) to determine whether failure to move during intentionally simulated and subjectively experienced paralysis is mediated by different neural structures. METHODS: Using a within-subject design, 12 normal, hypnotised subjects were tested under two paralysis conditions during the same scanning session. Half of the scans were performed with the suggestion that the left leg was paralysed (subjectively experienced paralysis condition) and half with the leg normal but with the instruction that paralysis should be feigned (intentionally simulated paralysis condition). RESULTS: Relative increases in brain activation were seen in the right orbitofrontal cortex, right cerebellum, left thalamus, and left putamen during subjectively experienced paralysis compared to intentionally simulated paralysis, although a previously reported activation of the right anterior cingulate cortex was not seen. During intentionally simulated paralysis compared to subjectively experienced paralysis relative increases in brain activation were seen in the left ventrolateral prefrontal cortex, and a number of right posterior cortical structures. CONCLUSIONS: Our results suggest that subjectively experienced paralysis has a different neural basis to intentionally simulated paralysis. These findings have theoretical and clinical implications for malingering and related attempts to unravel the neuropsychological basis for conversion hysteria.

The distribution of structural neuropathology in pre-clinical Huntington's disease.

Putative neuroprotective agents in Huntington's disease may have particular application before brain pathology becomes manifest clinically. If these agents were to be tested in clinical trials, a reliable marker of the burden and rate of progression of pathological change in the pre-clinical group would be needed. The present study investigates whether the Huntington's disease genotype is associated with regional differences in brain structure, particularly differences that could not be predicted from clinical or neuropsychological assessment. A secondary aim is to seek indirect evidence of pathological progression in the form of changes in local tissue volume with age, specific to the Huntington's disease genotype. Formal motor examination, neuropsychological assessment, and T(1)-weighted cerebral MRI were performed in 34 subjects who had undergone predictive genetic testing for Huntington's disease. Clinical and cognitive testing were performed blinded to gene status. A linear discriminant analysis revealed the combination of test scores (the 'optimal clinical score') which best differentiated 18 subjects carrying the Huntington's disease gene mutation (the 'gene-positive' group). Voxel-based morphometry (VBM) was used to identify regions of significant main effect of Huntington's disease gene status on grey and white matter volume and regions of significant interaction of gene status with age. In the gene-positive group, there was significant reduction in grey matter volume in the left striatum, bilateral insula, dorsal midbrain and bilateral intra-parietal sulcus relative to 'gene-negative' controls. There was a significant reduction of periventricular white matter volume with age bilaterally in the gene-positive relative to the gene-negative group. Changes remained significant when controlled for differences in optimal clinical score between subjects. This study provides evidence of distributed grey matter pathology and progressive white matter atrophy with age before clinical onset of Huntington's disease. This suggests that VBM may be useful in monitoring cross-sectional and longitudinal changes in brain structure in pre-clinical Huntington's disease and for determining the efficacy of neuroprotective agents.

Whose arm is it anyway? An fMRI case study of supernumerary phantom limb.

Under normal circumstances, information from a number of sources is combined to compute a unitary percept of the body. However, after pathology these influences may be perceived simultaneously, resulting in multiple dissociated conscious representations. In a recent paper, we described subject E.P., a right-handed female stroke patient with a right frontomesial lesion who sporadically experiences a supernumerary 'ghost' left arm that occupies the previous position of the real left arm after a delay of 60-90 s. We used a delayed response paradigm with functional MRI to examine the haemodynamic correlates of E.P.'s illusion. Comparison of periods of time during scanning when the ghost arm was present against when it was not revealed a single cluster (9 voxels, t = 5.11, P < 0.012 corrected for multiple comparisons) located on the right medial wall in the supplementary motor area ('SMA proper'). Our results suggest that areas traditionally classified as part of the motor system can influence the conscious perception of the body. We propose that, as a consequence of her injury, E.P. is aware of the position of the phantom limb in this 'action space' while also continuing to be aware of the true position of her real limb on the basis of afferent somatosensory information.

MRI analysis of an inherited speech and language disorder: structural brain abnormalities.

Analyses of brain structure in genetic speech and language disorders provide an opportunity to identify neurobiological phenotypes and further elucidate the neural bases of language and its development. Here we report such investigations in a large family, known as the KE family, half the members of which are affected by a severe disorder of speech and language, which is transmitted as an autosomal-dominant monogenic trait. The structural brain abnormalities associated with this disorder were investigated using two morphometric methods of MRI analysis. A voxel-based morphometric method was used to compare the amounts of grey matter in the brains of three groups of subjects: the affected members of the KE family, the unaffected members and a group of age-matched controls. This method revealed a number of mainly motor- and speech-related brain regions in which the affected family members had significantly different amounts of grey matter compared with the unaffected and control groups, who did not differ from each other. Several of these regions were abnormal bilaterally, including the caudate nucleus, which was of particular interest because this structure was also found to show functional abnormality in a related PET study. We performed a more detailed volumetric analysis of this structure. The results confirmed that the volume of this nucleus was reduced bilaterally in the affected family members compared with both the unaffected members and the group of age-matched controls. This reduction in volume was most evident in the superior portion of the nucleus. The volume of the caudate nucleus was significantly correlated with the performance of affected family members on a test of oral praxis, a test of non-word repetition and the coding subtest of the Wechsler Intelligence Scale. These results thus provide further evidence of a relationship between the abnormal development of this nucleus and the impairments in oromotor control and articulation reported in the KE family.

Anatomic constraints on cognitive theories of category specificity.

Many cognitive theories of semantic organization stem from reports of patients with selective, category-specific deficits for particular classes of objects (e.g., fruit). The anatomical assumptions underlying the competing claims can be evaluated with functional neuroimaging but the findings to date have been inconsistent and insignificant when standard statistical criteria are adopted. We hypothesized that category differences in functional brain responses might be small and task dependent. To test this hypothesis, we entered data from seven PET studies into a single multifactorial design which crossed category (living vs man-made) with a range of tasks. Reliable category-specific effects were observed but only for word retrieval and semantic decision tasks. Living things activated medial aspects of the anterior temporal poles bilaterally while tools activated a left posterior middle temporal region. These category-by-task interactions provide robust evidence for an anatomical double dissociation according to category and place strong constraints on cognitive theories of the semantic system. Furthermore they reconcile some of the apparent inconsistencies between lesion studies and functional neuroimaging data.