Glucocorticoid feedback sensitivity and adrenocortical responsiveness in posttraumatic stress disorder.

BACKGROUND: Decreased basal cortisol levels have been reported in individuals with posttraumatic stress disorder (PTSD). There is evidence for enhanced negative feedback sensitivity of the hypothalamic-pituitary-adrenal (HPA) axis in PTSD, which could account for this, but other possible mechanisms have not been ruled out. We examined the HPA axis employing a metyrapone-cortisol infusion protocol designed to study negative feedback sensitivity. METHODS: Vietnam combat trauma-exposed subjects met DSM-IV criteria for PTSD. Exclusion criteria included substance abuse and most medications. Endogenous feedback inhibition was removed by blocking cortisol synthesis with oral metyrapone and reintroduced by intravenous infusion of cortisol. In a placebo condition, subjects received oral placebo and normal saline infusion. Serial blood samples drawn over 4 hours were assayed for adrenocorticotrophic hormone (ACTH), cortisol, and 11-deoxycortisol. Selected samples were assayed for cortisol binding globulin (CBG) and dehydroepiandrosterone (DHEA). RESULTS: Basal plasma cortisol was significantly decreased in PTSD subjects (n = 13) compared with control subjects (n = 16). No significant difference in the ACTH response to cortisol infusion following metyrapone was observed; however 11-deoxycortisol was significantly decreased in PTSD subjects. In addition, CBG was significantly increased in PTSD subjects, and DHEA was significantly decreased in both PTSD and combat-exposed control subjects. CONCLUSIONS: These observations suggest decreased adrenocortical responsiveness may be an additional or alternative mechanism accounting for low cortisol in PTSD.

Biological markers and diagnostic accuracy in the genetics of posttraumatic stress disorder.

Family and twin studies suggest a substantial genetic contribution to the etiology of posttraumatic stress disorder (PTSD). Identification of the nature of this genetic contribution should enhance understanding of the pathophysiology of PTSD and suggest improved therapeutic strategies for its treatment. However, a broadly defined phenotype, specific requirement for an environmental exposure and high frequency of comorbid psychiatric illness all complicate genetic studies of PTSD. It is likely that genetic heterogeneity, incomplete penetrance, pleiotropy and the involvement of more than one gene all constitute formidable obstacles to the genetic analysis of PTSD. One way to circumvent these problems is to perform genetic analysis of traits associated with PTSD, rather than PTSD itself, an approach that has been fruitful for other diseases with complex modes of inheritance. Hypothalamic-pituitary-adrenal axis hypofunction, physiologic markers of increased arousal, and increased acoustic startle response are all potential PTSD-associated traits that might be susceptible to genetic analysis. However, the capacity of these traits to distinguish PTSD from non-PTSD patients and their familial pattern must be better defined before they can be employed in genetic studies.

Which duration of postsaccadic slowing identifies anticipatory saccades during smooth pursuit eye movements?

Increased frequency of anticipatory saccades during smooth pursuit eye movements is a potential marker of genetic risk for schizophrenia. Postsaccadic slowing criteria are used to separate anticipatory from other types of saccades. However, the necessary duration of slowed pursuit required to identify an anticipatory saccade remains undetermined. We explored the effect of various postsaccadic slowing duration criteria on effect size in a comparison of younger and older schizophrenic and normal adults. For large anticipatory saccades, varying the duration of postsaccadic slowing criteria did not notably change effect size. For smaller leading saccades, a limited 50-ms duration postsaccadic slowing criterion produced the largest effect size (1.54), and maintained a similar effect size across a broad age range. Leading saccades with a limited duration postsaccadic slowing criteria are a possible marker of genetic risk for schizophrenia.

Smooth pursuit eye movements in schizophrenia and attentional dysfunction: adults with schizophrenia, ADHD, and a normal comparison group.

BACKGROUND: Smooth pursuit eye movement (SPEM) abnormalities are found in schizophrenia. These deficits often are explained in the context of the attentional and inhibitory deficits central to schizophrenia psychopathology. It remains unclear, however, whether these attention-associated eye movement abnormalities are specific to schizophrenia or are a nonspecific expression of attentional deficits found in many psychiatric disorders. Adult attention-deficit/hyperactivity disorder (ADHD) is an alternative disorder with chronic attentional and inhibitory dysfunction. Thus, a comparison of SPEM in adult schizophrenia and adult ADHD will help assess the specificity question. METHODS: SPEM is recorded during a 16.7 degrees per second constant velocity task in 17 adults with ADHD, 49 adults with schizophrenia, and 37 normal adults; all groups included individuals between ages 25-50 years. RESULTS: Smooth pursuit gain and the frequency of anticipatory and leading saccades are worse in schizophrenic subjects, with normal and ADHD subjects showing no differences on these variables. CONCLUSIONS: Many attention-associated SPEM abnormalities are not present in most subjects with ADHD, supporting the specificity of these findings to the attentional deficits seen in schizophrenia.

Eye movement task measures inhibition and spatial working memory in adults with schizophrenia, ADHD, and a normal comparison group.

Schizophrenia and attention deficit/hyperactivity disorder (ADHD) are both associated with deficits in inhibition and working memory, although in ADHD the working memory deficit is hypothesized to be secondary to the inhibitory deficit. This similarity in cognitive processes has been paralleled by similarities across the two groups in the performance of working memory and inhibition tasks. The delayed oculomotor response task is an alternative task, which may allow greater separation of working memory from inhibitory components, and thus its use may provide additional information on primary vs. secondary deficits in these disorders. Ten young adult ADHD sufferers, 10 schizophrenic subjects, and 10 normal subjects were matched on age, gender, and education. Eye movements were recorded during delayed oculomotor response tasks with 1- and 3-s delays. Both the ADHD and the schizophrenic subjects demonstrated dis-inhibition (an increased percentage of premature saccades); however only schizophrenic subjects demonstrated an impaired working memory (decreased spatial accuracy of the remembered saccade). The results are consistent with the hypothesis that working memory is a primary deficit in schizophrenia, but secondary to the inhibitory deficit in ADHD.

The effects of age on a smooth pursuit tracking task in adults with schizophrenia and normal subjects.

BACKGROUND: Performance during a smooth pursuit eye movement (SPEM) task has been proposed as a marker of genetic risk for schizophrenia, although the precise component of SPEM tracking most associated with genetic risk remains undetermined. Normal adult aging is associated with deterioration on SPEM tasks; it remains unclear whether investigations of SPEM abnormalities will allow inclusion of older subjects in genetic studies. This study examines 1) the effect of normal aging on several components of SPEM performance; and 2) whether schizophrenic-normal differences found in young adults continue over a broad adult age span. METHODS: SPEM was recorded during a 16.7 degrees per sec constant velocity task in 64 normal adults, ages 18 to 79 years, and 58 schizophrenic subjects, ages 18 to 70 years. RESULTS: Smooth pursuit gain, the percent of total eye movements due to catch-up saccades, the frequency of large anticipatory saccades, and the frequency of leading saccades all deteriorate with increasing age. After correction for age, schizophrenic to control differences persist on most eye movement variables with the largest effect sizes for leading saccades (1.56) and smooth pursuit gain (1.17). CONCLUSIONS: The tendency to use saccades to anticipate target motion, even in small steps (leading saccades), deserves further attention as a potential marker useful in genetic analyses.

Amplitude criteria and anticipatory saccades during smooth pursuit eye movements in schizophrenia.

Increased frequency of anticipatory saccades during smooth pursuit eye movements is a potential marker of genetic risk for schizophrenia even in the absence of clinical symptomology. The operational definition of anticipatory saccades has often included an amplitude criterion; however, these amplitude criteria have often differed across studies. This study reports on the effect of varying amplitude criteria on the effect size in a comparison of 29 schizophrenic adults and 29 normal subjects during a 16.7 degrees/s constant velocity task. The inclusion of small amplitude anticipatory saccades, with amplitudes of 1-4 degrees, consistently increased effect size (largest effect size = 1.61). The inclusion of large anticipatory saccades, with amplitudes of 4 degrees or greater, had an inconsistent impact on effect size. The separation of anticipatory saccades into leading saccades (anticipatory saccades with amplitude 1-4 degrees) and large anticipatory saccades (amplitude > 4 degrees) deserves further exploration.

Evidence for bilineal inheritance of physiological indicators of risk in childhood-onset schizophrenia.

Childhood-onset schizophrenia is proposed to be associated with increased genetic loading compared with adult-onset schizophrenia because of its earlier age of onset and generally greater severity of symptoms. Diminished suppression of P50 auditory evoked responses to repeated stimuli and elevated anticipatory saccades during smooth pursuit eye movements are markers of genetic risk that are found in members of families with schizophrenia even in the absence of the full clinical disorder and appear to be transmitted in a single gene autosomal dominant fashion. Adult-onset schizophrenia is generally associated with one parent who demonstrates abnormal P50 sensory gating and elevated anticipatory saccades and one parent who is normal on the physiologic measures (i.e., unilineal inheritance). This study investigates whether childhood-onset schizophrenia is similarly unilineal or is associated with the inheritance of genetic risk factors from both parents (i.e., bilineal inheritance). Ten childhood-onset schizophrenic probands and their parents were studied. Their P50 sensory gating and anticipatory saccades were compared with adult-onset schizophrenic probands and their parents. Bilineality, measured as physiological impairment in both parents, occurred more frequently in childhood-onset probands than in adult-onset probands for both P50 sensory gating deficits (60% versus 13%) and elevated anticipatory saccades (60 versus 0%). Additionally, childhood-onset schizophrenic probands performed more poorly than adult-onset probands on the anticipatory saccade measure. This physiological evidence suggests that childhood-onset schizophrenia may be associated with increased genetic loading because of contributions of genetic risk from both parents.

Anticipatory saccades during smooth pursuit eye movements and familial transmission of schizophrenia.

BACKGROUND: Smooth pursuit eye movement (SPEM) abnormalities are a putative marker of genetic risk for schizophrenia. Accurate SPEM performance requires the subject to activate neural systems responsible for smooth pursuit tracking, while simultaneously suppressing activity of neurons responsible for saccadic movements that would move the eye ahead of the target. This study examined whether specific aspects of SPEM dysfunction cosegregate with genetic risk in parents of schizophrenic probands. METHODS: Eighteen probands and their parents had SPEM recorded. Parents with an ancestral history of schizophrenia were hypothesized to be more likely than their spouses without such a history to carry a genetic risk for schizophrenia. RESULTS: Ten families had a single parent with a positive ancestral history for schizophrenia. The frequency of anticipatory saccades, which were mostly small, and the fraction of total eye movement that they represented were the only measures that differentiated the more likely genetic carrier parents in these families from their spouses and age-matched normals. CONCLUSIONS: Failure to suppress saccadic anticipation of target motion during smooth pursuit appears an aspect of SPEM dysfunction related to presumed genetic risk for schizophrenia.

Does ketamine-mediated N-methyl-D-aspartate receptor antagonism cause schizophrenia-like oculomotor abnormalities?

Evidence from histological and pharmacological challenge studies indicates that N-methyl-D-aspartate (NMDA) receptor hypofunction may play an important role in the pathophysiology of schizophrenia. Our goal was to characterize effects of NMDA hypofunction further, as related to schizophrenia-associated neuropsychological impairment. We administered progressively higher doses of ketamine (target plasma concentrations of 50, 100, 150, and 200 ng/ml) to 10 psychiatrically healthy young men in a randomized, single-blind, placebo-controlled design and assessed oculomotor, cognitive, and symptomatic changes. Mean ketamine plasma concentrations approximated target plasma concentrations at each infusion step. Verbal recall, recognition memory, verbal fluency, pursuit tracking, visually guided saccades, and fixation all deteriorated significantly during ketamine infusion; lateral gaze nystagmus explained some, but not all, of the smooth pursuit abnormalities. We concluded that ketamine induces changes in recall and recognition memory and verbal fluency reminiscent of schizophreniform psychosis. During smooth pursuit eye tracking, ketamine induces nystagmus as well as abnormalities characteristic of schizophrenia. These findings help delineate the similarities and differences between schizophreniform and NMDA-blockade-induced cognitive and oculomotor abnormalities.

Familial transmission of two independent saccadic abnormalities in schizophrenia.

Difficulties with inhibiting inappropriate responses, i.e. disinhibition, and problems with spatial memory are both presumed to be a part of the phenotypic expression of the genetic risk for schizophrenia. Schizophrenic probands are impaired on saccadic eye movement tasks which require (a) response inhibition to prepotent stimuli and (b) generation of an accurate response to a remembered or calculated spatial location, but it is unknown how these deficits are inherited. Sixteen schizophrenic probands, their 32 parents, and two normal control groups completed a delayed oculomotor response and an antisaccade task. The parents with a positive ancestral family history for chronic psychosis (n = 8) were presumed to be more likely than their family history-negative spouses to be genetic carriers for schizophrenia. Probands and their positive family history parents had more failures of response inhibition than did normal control groups. However, it was the probands and their negative family history spouses who demonstrated impaired accuracy of the remembered- or antisaccades. Disinhibition may be closely tied to a specific genetic risk for schizophrenia. However, a second familial factor related to the maintenance or manipulation of spatial information may also contribute to the genetic risk of the full clinical disorder.

Psychedelic effects of ketamine in healthy volunteers: relationship to steady-state plasma concentrations.

BACKGROUND: Ketamine has been associated with a unique spectrum of subjective "psychedelic" effects in patients emerging from anesthesia. This study quantified these effects of ketamine and related them to steady-state plasma concentrations. METHODS: Ketamine or saline was administered in a single-blinded crossover protocol to 10 psychiatrically healthy volunteers using computer-assisted continuous infusion. A stepwise series of target plasma concentrations, 0, 50, 100, 150, and 200 ng/ml were maintained for 30 min each. After 20 min at each step, the volunteers completed a visual analog (VAS) rating of 13 symptom scales. Peripheral venous plasma ketamine concentrations were determined after 28 min at each step. One hour after discontinuation of the infusion, a psychological inventory, the hallucinogen rating scale, was completed. RESULTS: The relation of mean ketamine plasma concentrations to the target concentrations was highly linear, with a correlation coefficient of R = 0.997 (P = 0.0027). Ketamine produced dose-related psychedelic effects. The relation between steady-state ketamine plasma concentration and VAS scores was highly linear for all VAS items, with linear regression coefficients ranging from R = 0.93 to 0.99 (P < 0.024 to P < 0.0005). Hallucinogen rating scale scores were similar to those found in a previous study with psychedelic doses of N,N-dimethyltryptamine, an illicit LSD-25-like drug. CONCLUSIONS: Subanesthetic doses of ketamine produce psychedelic effects in healthy volunteers. The relation between steady-state venous plasma ketamine concentrations and effects is highly linear between 50 and 200 ng/ml.

Relationships between neuropsychological and oculomotor measures in schizophrenia patients and normal controls.

Establishing the relationship between oculomotor and neuropsychological impairments might facilitate a more coherent description of schizophrenia-associated neurocognitive deficits. Therefore, we assessed several aspects of neuropsychological and oculomotor function in 25 medicated schizophrenia patients and 24 age-matched controls. Neuropsychological tasks included the Wisconsin Cart Sort Test (WCST), the Trail Making Test (TMT), the Rey Auditory Verbal Learning Test, and finger tapping speed. Oculomotor functions assessed included smooth pursuit, initiation of smooth pursuit, predictive pursuit, fixation, visually guided saccades, remembered saccades, and antisaccades. Among the schizophrenia patients, predictive pursuit performance correlated significantly with finger tapping (dominant hand), TMT (both parts), and one WCST measure (categories completed). The only other significant correlation among the schizophrenia patients was between antisaccade performance and part A of the TMT. Perseverative errors during the WCST and antisaccade performance were the only measures significantly correlated among the normals. Closely related neurocognitive deficits may be responsible for impairments in TMT, WCST, predictive pursuit, and antisaccade performance in schizophrenia.

A quantitative analysis of smooth pursuit eye tracking in monozygotic twins discordant for schizophrenia.

BACKGROUND: Previous studies of discordant monozygotic (MZ) twins have suggested that abnormal smooth pursuit eye tracking is an indicator of genetic liability for schizophrenia. We attempted to replicate this in a different sample of twins. METHODS: Probands from 12 sets of MZ twins discordant for schizophrenia who met DSM-III-R criteria for schizophrenia or schizoaffective disorder and their co-twins without psychiatric diagnosis (except 2 with a history of substance abuse) and 12 sets of normal control MZ twins. Psychiatric diagnosis was based on Structured Clinical Interview; monozygosity was based on analysis of 19 red blood cell antigens. Smooth pursuit eye movement gain (equal to the ratio of eye-target velocity) and numbers, amplitudes, and subtypes of saccadic eye movements were compared. Measures were derived from computer analysis of digitized infrared oculographic recordings of constant velocity (16.67 degrees per second) smooth pursuit eye tracking. RESULTS: Quantitative measures of eye tracking for the affected twin were inferior to those of the unaffected co-twin, with affected twins showing significant decreases in gain and significant increases in numbers and amplitudes of total and intrusive saccades. Moreover, whereas means for the group of affected twins differed significantly from those of normal controls on measures of gain and total saccades, means for the group of unaffected co-twins were well within the normal range. CONCLUSIONS: These data are consistent with the hypothesis that abnormal eye tracking is associated with the expression of illness, or phenotype, in schizophrenia, at least in this twin sample. The data raise questions regarding the use of eye tracking measurement for identifying putative gene carriers among at-risk relatives in genetic linkage studies of schizophrenia.

Reduced benzodiazepine sensitivity in patients with panic disorder: comparison with patients with obsessive-compulsive disorder and normal subjects.

OBJECTIVE: The authors sought to replicate their previous finding of reduced response to diazepam in patients with panic disorder, to test whether this effect was specific for panic disorder, and to determine whether this reduced response was merely an artifact of resistance to sedation from anxiety-related overarousal. METHOD: The effects of four increasing intravenous doses of diazepam on saccadic eye movement velocity and accuracy (the latter being a saccadic variable that is unaffected by sedation), short-term memory, and self- and observer-rated sedation were assessed in 18 patients with panic disorder, 15 patients with obsessive-compulsive disorder, and 14 normal comparison subjects. The ratios of effect to blood level areas under the curve for both ascending and descending limbs of the effect/blood level curves were compared for each variable. RESULTS: Patients with panic disorder showed significantly less diazepam effect on saccadic velocity and accuracy for the ascending limb of the blood level curve than comparison subjects. Patients with obsessive-compulsive disorder showed similar differences from comparison subjects but only for saccadic velocity. There were no group differences in diazepam effects on memory and sedation. CONCLUSIONS: Patients with panic disorder are less sensitive than comparison subjects to diazepam. Although this difference is not an artifact of resistance to sedation, it may not be specific for panic disorder but rather may reflect a more nonspecific aspect of anxiety disorders.

Early expression of smooth-pursuit eye movement abnormalities in children of schizophrenic parents.

OBJECTIVE: Disordered smooth-pursuit eye movements (SPEM) and, specifically, small anticipatory saccades that disrupt SPEM have been hypothesized to be a marker of genetic vulnerability to schizophrenia. This study compares SPEM in children of schizophrenic parents with normally developing control children to assess whether SPEM abnormalities are also present in a subset of at-risk children. METHOD: With infrared oculography, SPEM was examined in 13 children of schizophrenic parents and 19 normally developing controls (aged 6 to 15 years). Measures of smooth-pursuit gain and root mean square error were used in addition to more specific measures of catch-up saccades and anticipatory saccades. RESULTS: Children of schizophrenic parents differed from normally developing controls on gain and root mean square error, but not on catch-up saccades. Small anticipatory saccades were significantly more frequent in the at-risk group. The percentage of total eye movements due to anticipatory saccades identified 54% of the at-risk group (compared with none of the control group) as performing more than two standard deviations above (worse than) the control mean. CONCLUSIONS: The presence of increased anticipatory saccades is evidence for an oculomotor dysfunction that may be a phenotype of the genetic risk for schizophrenia, expressed years prior to the possible development of clinical illness.

Human oculomotor function: reliability and diurnal variation.

To provide information on test-retest reliability for seven oculomotor paradigms currently used in studies of schizophrenia and other neuropsychiatric conditions, we tested eight controls at four weekly intervals, twice in the morning (8-10 AM) and twice in the afternoon (3-5 PM). Intraclass correlation coefficients were significant (p < .05) for both AM and PM pairs of measures as well as for mean AM and PM pairs for closed-loop pursuit gain, open-loop pursuit gain (using velocity as the measure), saccadic frequency during pursuit and fixation, visually and nonvisually guided saccadic latency and velocity, antisaccadic latency, and premature reflexive saccades during the memory-guided saccade task. Acceleration as a measure of open-loop gain (for slower targets) and accuracy of saccades to a moving target were only reliable at PM testing time. Nonvisually guided saccadic accuracy and inappropriate reflexive saccades during the antisaccade task were not reliable, possibly due to the narrow range of values for these measures. Except for approximately 10% fewer saccades during pursuit and fixation in the morning, there were no consistent diurnal differences. These findings suggest that, in a small sample of subjects, most measures of oculomotor function are stable across time and may reflect underlying neurophysiologic traits.

Benzodiazepine sensitivity in panic disorder: effects of chronic alprazolam treatment.

The aim of the current study was to determine the degree to which patients with panic disorder develop tolerance to subjective and physiological effects of benzodiazepine after chronic treatment with alprazolam. Response to acute administration of diazepam was assessed in 19 panic disorder patients receiving chronic treatment with alprazolam and 23 untreated panic disorder patients. At baseline in the laboratory, the two groups did not differ in peak saccadic eye movement velocity, saccade latency, short-term memory, plasma cortisol and growth hormone concentrations, heart rate, and self-rated levels of sedation and anxiety. Compared with untreated patients, alprazolam-treated patients displayed significantly less diazepam-induced change in peak saccadic velocity, saccade latency, growth hormone secretion, memory, and self-rated levels of sedation. There was no difference between groups in diazepam effects on plasma cortisol concentrations or self-rated anxiety. Within alprazolam-treated patients, diazepam-induced slowing of peak saccade velocity was significantly inversely correlated with illness severity, as measured by reported panic attacks per week and severity of phobic avoidance, but not with alprazolam dose, blood level, or duration of treatment. Because the alprazolam-treated group reported more panic attacks per week than the untreated panic patients, treated patients were divided into those who were asymptomatic versus those with continuing panic attacks. The subgroup of nine alprazolam-treated subjects who were asymptomatic also showed significantly less diazepam effects than the group of untreated panic disorder patients, suggesting that overall group differences were at least partially attributable to the development of tolerance to selected benzodiazepine effects with chronic alprazolam treatment.

Effects of diphenhydramine on human eye movements.

Peak saccadic eye movement velocity (SEV) and average smooth pursuit gain (SP) are reduced in a dose-dependent manner by diazepam and provide reliable, quantitative measures of benzodiazepine agonist effects. To evaluate the specificity of these eye movement effects for agents acting at the central GABA-benzodiazepine receptor complex and the role of sedation in benzodiazepine effects, we studied eye movement effects of diphenhydramine, a sedating drug which does not act at the GABA-benzodiazepine receptor complex. Ten healthy males, aged 19-28 years, with no history of axis I psychiatric disorders or substance abuse, received 50 mg/70 kg intravenous diphenhydramine or a similar volume of saline on separate days 1 week apart. SEV, saccade latency and accuracy, SP, self-rated sedation, and short-term memory were assessed at baseline and at 5, 15, 30, 45, 60, 90 and 120 min after drug administration. Compared with placebo, diphenhydramine produced significant SEV slowing, and increases in saccade latency and self-rated sedation. There was no significant effect of diphenhydramine on smooth pursuit gain, saccade accuracy, or short-term memory. These results suggest that, like diazepam, diphenhydramine causes sedation, SEV slowing, and an increase in saccade latency. Since the degree of diphenhydramine-induced sedation was not correlated with changes in SEV or saccade latency, slowing of saccadic eye movements is unlikely to be attributable to sedation alone. Unlike diazepam, diphenhydramine does not impair smooth pursuit gain, saccadic accuracy, or memory. Different neurotransmitter systems may influence the neural pathways involved in SEV and smooth pursuit again.

Saccadic eye movements in normal children from 8 to 15 years of age: a developmental study of visuospatial attention.

This cross-sectional study used saccadic eye movements, as measured by infrared occulography, to assess several aspects of visuospatial attention in normal children ages 8-15 years. Saccadic latency (a global measure of the ability to shift visuospatial attention), the ability to suppress extraneous saccades during fixation, and the ability to inhibit task-provoked anticipatory saccades all improve with age. However, the pattern of development differs for different tasks; saccadic latency shortens at a linear rate across the age range 8-15 years, while the capacity to inhibit anticipatory saccades matures by 12-13 years of age, and the ability to suppress saccades matures by 10 years of age. Analyses of age-related changes in oculomotor measures of attention may provide a novel approach in the study of children with attentional difficulties.