Optimization of capture-recapture monitoring of elusive species illustrated with a threatened grasshopper.

Information on population sizes and trends of threatened species is essential for their conservation, but obtaining reliable estimates can be challenging. We devised a method to improve the precision of estimates of population size obtained from capture-recapture studies for species with low capture and recapture probabilities and short seasonal activity, illustrated with population data of an elusive grasshopper (Prionotropis rhodanica). We used data from 5 capture-recapture studies to identify methodological and environmental factors affecting capture and recapture probabilities and estimates of population size. In a simulation, we used the population size and capture and recapture probability estimates obtained from the field studies to identify the minimum number of sampling occasions needed to obtain unbiased and robust estimates of population size. Based on these results we optimized the capture-recapture design, implemented it in 2 additional studies, and compared their precision with those of the nonoptimized studies. Additionally, we simulated scenarios based on thresholds of population size in criteria C and D of the International Union for Conservation of Nature (IUCN) Red List to investigate whether estimates of population size for elusive species can reliably inform red-list assessments. Identifying parameters that affect capture and recapture probabilities (for the grasshopper time since emergence of first adults) and optimizing field protocols based on this information reduced study effort (-6% to -27% sampling occasions) and provided more precise estimates of population size (reduced coefficient of variation) compared with nonoptimized studies. Estimates of population size from the scenarios based on the IUCN thresholds were mostly unbiased and robust (only the combination of very small populations and little study effort produced unreliable estimates), suggesting capture-recapture can be considered reliable for informing red-list assessments. Although capture-recapture remains difficult and costly for elusive species, our optimization procedure can help determine efficient protocols to increase data quality and minimize monitoring effort.

Optimización del Monitoreo de Captura y Recaptura de Especies Esquivas Ilustrado con un Saltamontes Amenazado Resumen La información sobre los tamaños poblacionales y las tendencias de las especies amenazadas es esencial para su conservación, pero la obtención de estimaciones confiables puede ser todo un reto. Diseñamos un método para mejorar la precisión de las estimaciones del tamaño poblacional obtenidos de estudios de captura y recaptura para especies con probabilidades bajas de captura y recaptura y una corta actividad estacional y lo ilustramos con los datos poblacionales de un saltamontes esquivo (Prionotropis rhodanica). Usamos los datos de cinco estudios de captura y recaptura para identificar los factores metodológicos y ambientales que afectan a la probabilidad de captura y recaptura y a los estimados de tamaños poblacionales. En una simulación, usamos el tamaño poblacional y las estimaciones de probabilidad de captura y recaptura obtenidos en estudios de campo para identificar el número mínimo de ocasiones de muestreo necesarias para obtener estimaciones imparciales y sólidos del tamaño poblacional. Con base en estos resultados, optimizamos el diseño de la captura y recaptura, la implementamos en dos estudios adicionales y comparamos su precisión con aquella de los estudios no optimizados. Además, simulamos escenarios con base en los umbrales de tamaño poblacional localizados en los criterios C y D de la Lista Roja de la Unión Internacional para la Conservación de la Naturaleza (UICN) para conocer si las estimaciones del tamaño poblacional para especies esquivas pueden informar certeramente las valoraciones de lista roja. La identificación de los parámetros que afectan las probabilidades de captura y recaptura (desde el momento de aparición de los primeros saltamontes adultos) y la optimización de los protocolos de campo con base en esta información redujeron el esfuerzo de estudio (−6% a −27% ocasiones de muestreo) y proporcionaron estimaciones más precisas del tamaño poblacional (coeficiente reducido de variación) en comparación con los estudios no optimizados. Las estimaciones del tamaño poblacional tomadas de los escenarios basados en los umbrales de la UICN fueron, en su mayoría, imparciales y sólidos (sólo la combinación de poblaciones muy pequeñas y un esfuerzo mínimo de estudio produjo estimaciones no confiables), lo que sugiere que la captura y recaptura puede considerarse como confiable para informar las valoraciones de lista roja. Aunque la captura y recaptura todavía es complicada y costosa cuando se aplica a especies esquivas, ésta puede ayudar a determinar los protocolos eficientes para incrementar la calidad de los datos y minimizar el esfuerzo de monitoreo.

Hypothalamo-pituitary-adrenal axis, glucose metabolism and TNF-α in narcolepsy.

Narcolepsy with cataplexy is caused by a deficiency in the production of hypocretin/orexin, which regulates sleep and wakefulness, and also influences appetite, neuroendocrine functions and metabolism. In this case-control study, 11 patients with narcolepsy with cataplexy and 11 healthy adults underwent an oral glucose tolerance test, and dexamethasone suppression/corticotropin-releasing hormone stimulation test. The average age of patients and controls was 35.1 ± 13.2 and 41.0 ± 2.9 years, respectively, body mass index was 28.1 ± 6.6 and 25.5 ± 4.7 kg m(-2) . We did not find evidence of a significantly increased prevalence of disturbed glucose tolerance in patients with narcolepsy. After hypothalamo-pituitary-adrenal axis suppression, the number of non-suppressors did not differ between the groups, indicating normal negative feedback sensitivity. The level of cortisol after dexamethasone suppression was significantly lower in patients with narcolepsy, suggesting a slight basal downregulation and/or a slightly increased negative feedback sensitivity of the major endocrine stress system in narcolepsy. Following corticotropin-releasing hormone stimulation, there were no significant differences in levels of adrenocorticotropic hormone or cortisol, and in adrenocortical responsivity to adrenocorticotropic hormone. Finally, patients with narcolepsy displayed significantly higher plasma levels of tumour necrosis factor alpha, soluble tumour necrosis factor receptor p55, soluble tumour necrosis factor receptor p75 and interleukin 6 after adjustment for body mass index. The present study confirms that narcolepsy by itself is not associated with disturbances of glucose metabolism, but goes along with a subtle dysregulation of inflammatory cytokine production. We also found that dynamic hypothalamo-pituitary-adrenal system response is not altered, whereas negative feedback to dexamethasone might be slightly enhanced.

Effects of clozapine and olanzapine on cytokine systems are closely linked to weight gain and drug-induced fever.

The second generation antipsychotics clozapine and olanzapine are known to cause weight gain. However, only clozapine is associated with drug-induced fever. Cytokines have been linked to the induction of both weight gain and drug-induced fever. We investigated these potential side effects of clozapine and olanzapine and studied their differential effects on cytokine secretion. Thirty patients suffering from schizophrenia, schizophreniform disorder or schizoaffective disorder were treated with either clozapine (mean modal dose: 266.7+/-77.9mg) or olanzapine (21.2+/-2.5mg) in a randomized, double-blind, 6-week study. Body mass index (BMI), tympanic temperature, and plasma levels of leptin and cytokines (tumor necrosis factor-alpha (TNF-alpha), soluble TNF receptor 1 and 2 (sTNFR-1/2), soluble interleukin-2 receptors (sIL-2R), interleukin-6) were determined weekly. BMI, leptin and cytokines significantly increased over time, except interleukin-6 and sTNFR-1 in the olanzapine group. All cytokines numerically increased compared to baseline already during the first week of treatment in both groups. Leptin, TNF-alpha, sTNFR-1, sTNFR-2 and sIL-2R levels correlated with the BMI. Five patients who received clozapine (33%) developed drug-induced fever (>/=38 degrees C). In these, interleukin-6 peak levels were significantly (p<0.01) higher than in those patients treated with clozapine who did not develop fever. In conclusion, increase of BMI appears to be related to clozapine's and olanzapine's similar effects on cytokine systems, whilst drug-induced fever appears to be related to clozapine's differential effects on interleukin-6.

Immune-endocrine host response to endotoxin in major depression.

OBJECTIVE: An impaired hypothalamic-pituitary-adrenocortical (HPA) function is a well-established finding in major depression (MD), but it is still unclear how this dysfunction affects immune responses in this disorder. METHOD: To further examine the relationship between immune and endocrine responses in MD, 0.4ng/kg body weight endotoxin [LPS] or 100mug hCRH were sequentially applied to 12 patients with MD and to 12 age- and gender-matched healthy controls after pre-treatment with 1.5mg dexamethasone (DEX). Immune (TNF-alpha, IL-6, rectal temperature) and endocrine (ACTH, cortisol) parameters were examined as area under the curve (AUC) levels. RESULTS: After pre-treatment with DEX, LPS evoked an immune response in all participants of the study with most immune parameters significantly related to the endotoxin challenge. However, only a marked immune response resulted in an additional endocrine reaction. Subsequently, the quantitative extent of the endocrine reaction was related to the extent of the immune response after DEX/LPS challenge. Pre-LPS AUC levels of cortisol, ACTH and post-LPS levels of IL-6 as well as the post-CRH AUC levels of cortisol and ACTH were related to the depressive symptomatology as measured by the Beck depression inventory (BDI). In depressive patients who showed increased cortisol plasma levels before LPS, the later increase in IL-6 was reduced. CONCLUSIONS: The challenge with DEX/LPS did not reveal major impairments of evoked immune functions in MD. Only the endocrine parameters and the IL-6 response were related to the depressive symptomatology, suggesting a limited interaction between immune and endocrine dysfunctions in MD.

Successful antidepressant therapy restores the disturbed interplay between TNF-alpha system and HPA axis.

BACKGROUND: In depressed patients, alterations in the hypothalamo-pituitary-adrenocortical (HPA) system are the most consistent neurobiological finding. HPA axis activity and cytokines are intrinsically intertwined: inflammatory cytokines stimulate adrenocorticotropic hormone (ACTH) and cortisol secretion, while, in turn, glucocorticoids suppress the synthesis of proinflammatory cytokines. METHODS: We examined alterations in plasma levels of tumor necrosis factor-alpha (TNF-alpha), levels of its soluble receptors p55 (sTNF-R p55) and p75 (sTNF-R p75) as well as changes in the HPA system function using the combined dexamethasone/corticotropin-releasing hormone (dex/CRH) test on admission and at discharge in 70 depressed inpatients without inflammation. RESULTS: On admission, TNF-alpha levels were inversely associated with the ACTH response to the combined dex/CRH test. Changes in TNF-alpha, sTNF-R p55, and sTNF-R p75 plasma levels from admission to discharge were positively correlated with the dex/CRH test outcome at discharge. Subgroup analysis revealed that this association was restricted to those patients achieving remission. In this subgroup, TNF-alpha levels at discharge were also positively correlated with dex/CRH test response at discharge. CONCLUSIONS: Our results suggest that elevated HPA axis activity in acute depression suppresses TNF-alpha system activity, while after remission, when HPA axis activity has normalized, the TNF-alpha system seems to gain influence on the HPA system.

Plasma levels of leptin and endogenous immune modulators during treatment with carbamazepine or lithium.

Several psychopharmacological agents induce weight gain. Recent studies have suggested that the tumor necrosis factor-alpha (TNF-alpha) cytokine system is pathophysiologically involved. To assess whether carbamazepine and lithium, which have been reported to lead to weight gain, have effects on the circulating levels of cytokines, we measured plasma levels of TNF-alpha, its soluble receptors sTNF-R p55 and p75, and leptin, as well as weight in 25 inpatients receiving lithium (n=10) or carbamazepine (n=15) weekly during the first 4 weeks of treatment. We found an increase in the body mass index and in TNF-alpha and its soluble receptor levels, but not in leptin levels over the 4 weeks of treatment. These changes did not differ between treatment groups. Changes of weight during the first week of treatment, but no other parameter, strongly predicted weight change until endpoint. We conclude that the mood stabilizers carbamazepine and lithium have similar effects on the TNF-alpha system and do not affect leptin levels.

Elevation of liver enzyme levels during psychopharmacological treatment is associated with weight gain.

Increased circulating levels of liver enzymes emerging during treatment with psychotropic drugs are frequently encountered and, in general, attributed to drug metabolism or toxic effects. Because obesity was shown to be associated with elevated liver enzyme levels in different non-psychiatric study samples, we hypothesized that drug-induced weight gain might be an additional causative factor. We tested this hypothesis in 67 inpatients who received psychopharmacological treatment across five weeks. Stepwise linear regression was used to predict changes in the serum levels of aspartate-amino transferase (ASAT) and alanine-amino transferase (ALAT) by changes in the body mass index (BMI), by changes in other biological parameters related to body weight (tumor necrosis factor-alpha [TNF-alpha], soluble TNF receptors [sTNF-R], interleukin-6 [IL-6], leptin plasma levels) and by the respective liver enzyme baseline level. BMI changes from baseline to endpoint were significantly associated with the changes in ALAT and ASAT levels across five weeks of treatment and with ALAT and ASAT levels at the end point of the study. The baseline levels of ALAT and ASAT also had a significant impact on these liver enzyme level changes, whereas all other variables had not. These results suggest that weight gain-associated metabolic changes occurring during treatment with psychotropic drugs have consistent and clinically relevant effects on the liver.

Reduced cortical gray matter in narcolepsy: preliminary findings with voxel-based morphometry.

There is a selective loss of hypocretin/orexin-containing hypothalamic neurons in patients with narcolepsy. The authors compared MRI-derived gray matter maps of 12 patients with narcolepsy with matched controls using voxel-based morphometry to ascertain whether there are other structural brain abnormalities. Patients with narcolepsy showed bilateral cortical gray matter reductions predominantly in inferior temporal and inferior frontal brain regions. Relative global gray matter loss was independent of disease duration or medication history. No significant subcortical gray matter alterations were noted.

An automatic method for scoring leg movements in polygraphic sleep recordings and its validity in comparison to visual scoring.

STUDY OBJECTIVES: The study presents an automatic method for scoring leg movements in polysomnographic recordings and describes an empirical investigation of its validity. DESIGN: Leg movements measured by means of the surface electromyogram activation of the right and left tibialis anterior muscle contained in 24 digitally recorded all-night polysomnograms were analyzed visually according to the American Sleep Disorders Association guidelines by 2 experienced raters and automatically scored using a newly developed electromyogram-based analytical method. Two visual scorings and the automatic scoring were compared in pairs using descriptive and confirmative statistical methods. SETTING: N/A. PARTICIPANTS: All-night polysomnograms of adaptation nights at the sleep laboratory of 10 patients with idiopathic restless legs syndrome (RLS) according to the International RLS Study Group. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Agreement rates between the 2 well-trained scorers and the automatic method were comparable. Based on the first scorer's results (100%) 92.5% of the movements were detected by the second scorer and 94.3% by the automatic method. When the visual scorings were compared, the rate of false-positive and false-negative errors were 7% and 3%, respectively. Comparing both visual scorings with the results of the automatic scoring yielded false-positive and false-negative rates in the range from 3% to 8%. CONCLUSIONS: The degree of accordance between the 2 visual scorings and between the visual and the automatic scorings were comparable. Therefore, this method is valid and may be used for the automatic detection of leg movements in future studies.

Diurnal variations of interleukin-6 plasma levels are confounded by blood drawing procedures.

Recent findings suggest that inflammatory cytokines are involved in sleep regulation. In part, this idea is based on studies showing that systemic levels of interleukin-6 (IL-6) are affected by sleep and sleep deprivation. However, intravenous (IV) catheters used for repetitive blood sampling were reported to increase local IL-6 production, which might confound sleep-dependent or circadian changes in the plasma concentrations of this cytokine. To further examine the effects of blood drawing procedures on IL-6 plasma levels, 12 healthy young male subjects participated in a 24-h cross-over study protocol involving sleep and sleep deprivation. Blood was collected half-hourly through an IV line and one additional sample was taken by a simple needle stick from the contralateral arm in parallel to the last sample from the catheter. Difficulties in blood sampling, the plasma levels of IL-6, cortisol and subjective sleepiness were quantified. In samples from the IV line there was a linear increase in IL-6 levels in both conditions, whereas the amount of IL-6 detected in the needle stick sample at the end did not differ from baseline. IL-6 levels were significantly higher in samples rated as difficult and those difficulties were more frequent during sleep compared to nocturnal wakefulness. IL-6 levels did not correlate to variations in sleepiness or cortisol levels. We conclude that variations in IL-6 plasma levels measured in samples from an IV catheter are caused, at least in part, by changes in local cytokine production rather than by physiological changes in circulating IL-6 levels.

Early prediction of changes in weight during six weeks of treatment with antidepressants.

Weight gain is a frequent and important side effect of psychotropic treatment. We sought to determine weight change predictors during treatment with antidepressant drugs. In 24 patients weight, plasma levels of leptin, tumor necrosis factor-alpha (TNF-alpha and soluble TNF receptors were determined longitudinally and a multiple linear regression analysis was used to predict weight change from baseline to the sixth week of treatment. Changes of weight during the first week of treatment, but no other parameter, strongly predicted weight change until endpoint (adjusted R2 = 0.68, F = 50.45, df = 1,23, p < 0.001). Very early changes in weight during treatment with psychotropic drugs might be a simple and clinically useful predictor of future weight development.

Epidemiology, implications and mechanisms underlying drug-induced weight gain in psychiatric patients.

Body weight gain frequently occurs during drug treatment of psychiatric disorders and is often accompanied by increased appetite or food craving. While occurrence and time course of this side effect are difficult to predict, it ultimately results in obesity and the morbidity associated therewith in a substantial part of patients, often causing them to discontinue treatment even if it is effective. This paper reviews the available epidemiological data on the frequency and extent of weight gain associated with antidepressant, mood-stabilizing, and antipsychotic treatment. Possible underlying pathomechanisms are discussed with special attention to central nervous control of appetite including the role of leptin and the tumor necrosis factor system. Metabolic alterations induced by drug treatment such as type 2 diabetes mellitus and the metabolic syndrome are also considered. Weight gain appears to be most prominent in patients treated with some of the second generation antipsychotic drugs and with some mood stabilizers. Marked weight gain also frequently occurs during treatment with most tricyclic antidepressants, while conventional antipsychotics typically induce only slight to moderate weight gain. Serotonin reuptake inhibitors may induce weight loss during the first few weeks, but some of them induce weight gain during long-term treatment. Several antidepressant and antipsychotic drugs are identified which reliably do not cause weight gain or even reduce weight. Based on these insights, countermeasures to manage drug-induced weight gain are suggested.

Hypothalamo-pituitary-adrenal function in patients with depressive disorders is correlated with baseline cytokine levels, but not with cytokine responses to hydrocortisone.

Dysfunction of the hyopthalamo-pituitary adrenal (HPA) system is frequently found in major depression. In addition, signs of non-specific inflammatory system activation have been reported. However, very little is known about interactions between the HPA and immune systems in depressive patients. To assess HPA system function, we performed a combined dexamethasone suppression and corticotropin-releasing hormone stimulation (DEX/CRH) test in 14 depressive patients. Moreover, baseline nocturnal plasma levels of the inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor (TNF)-alpha were measured. In addition, the system was challenged with an intraveneous pulsatile injection of hydrocortisone (1 mg/kg body weight in total) and again cytokine levels were measured across one night. Baseline TNF-alpha levels were negatively correlated with the amount of ACTH released upon CRH stimulation during the DEX/CRH test. Acute hydrocortisone administration suppressed TNF-alpha and IL-6 levels independently of baseline HPA system activity. We conclude that chronic HPA system overactivity in depressed patients might compromise the production of inflammatory cytokines under baseline conditions. However, the responsivity of the cytokine production to acutely administered glucocorticoids does not seem to correlate with the state of the HPA system.

Increased body mass index (BMI) in male narcoleptic patients, but not in HLA-DR2-positive healthy male volunteers.

BACKGROUND: There is growing evidence that hypocretin deficiency plays a pivotal role in human narcolepsy. Based on the physiological role of hypocretins in the regulation of food intake, one might suspect that narcoleptic patients should display reduced energy intake and as a consequence a reduced body weight compared to healthy controls. METHODS: The body mass indices (BMIs) of 30 male narcoleptic patients were compared with large community-based reference samples. Because it is unclear whether increased BMI is an acquired consequence of the disease or a genetically determined premorbid feature of narcolepsy, we additionally examined the influence of the HLA-DR2 antigen, strongly associated with narcolepsy, on the BMI in a group of 117 healthy male volunteers. RESULTS: Narcoleptic patients displayed higher age- and gender-specific BMI percentiles compared to a community-based sample from the German (normal) population. Within the patient sample, BMI distribution did not significantly differ between subjects who had previously received pharmacological treatment compared to drug-naive patients, or between the HLA-DR2 positive and negative healthy subjects. CONCLUSIONS: The results of the present study suggest that an increased BMI in narcolepsy is neither associated with the HLA-DR2 antigen per se nor with medication, but is more likely to be a consequence of disease-associated neuroendocrine abnormalities.

Olanzapine and clozapine differently affect sleep in patients with schizophrenia: results from a double-blind, polysomnographic study and review of the literature.

Schizophrenia is associated with impaired sleep continuity. The second generation antipsychotics clozapine and olanzapine have been reported to improve sleep continuity but also to rarely induce restless legs syndrome (RLS). The aims of this randomized double-blind study were to compare the effects of clozapine and olanzapine on sleep and the occurrence of RLS. Therefore, polysomnographies were recorded and RLS symptoms were assessed in 30 patients with schizophrenia before and after 2, 4 and 6 weeks of treatment with either clozapine or olanzapine. Treatment with both antipsychotics increased total sleep time, sleep period time and sleep efficiency and decreased sleep onset latency. These changes were similar in both groups, occurred during the first 2 treatment weeks and were sustained. For example, sleep efficiency increased from 83% (olanzapine) and 82% (clozapine) at baseline to 95% at week 2 and 97% at week 6 in both treatment groups. Sleep architecture was differently affected: clozapine caused a significantly stronger increase of stage 2 sleep (44%) than olanzapine (11%) but olanzapine a significantly stronger increase of REM-sleep. Olanzapine caused an 80% increase of slow wave sleep whereas clozapine caused a 6% decrease. No patient reported any of 4 RLS defining symptoms at baseline. During treatment, 1 patient of each group reported at one visit all 4 symptoms, i.e. met the diagnosis of an RLS. In conclusion, sleep continuity similarly improved and sleep architecture changed more physiologically with olanzapine. Neither of the antipsychotics induced RLS symptoms that were clinically relevant.

Glucose tolerance in patients with narcolepsy.

STUDY OBJECTIVES: Obesity is a common feature of narcolepsy. In addition, an increased occurrence of non-insulin dependent diabetes has been reported. So far, it is not known whether glucose metabolism in narcolepsy is disturbed due to, or independently of obesity. DESIGN: Case-control study. SETTING: Sleep medicine clinic at a research institute. PATIENTS: We studied 17 patients with narcolepsy/cataplexy compared to 17 healthy controls matched for age, sex, and body mass index (BMI). INTERVENTIONS: A 75-g oral glucose tolerance test was performed. MEASUREMENTS: Glucose tolerance was determined by computing plasma glucose curve following oral glucose challenge for 240 minutes; insulin sensitivity and insulin secretion by homeostasis model assessment and minimal model analysis. RESULTS: Standard outcome measures and indices of the oral glucose tolerance test did not differ between the patient group and the group of control subjects. CONCLUSIONS: In this study, no clinically relevant pathologic findings in the glucose metabolism of narcoleptic patients compared to weight matched controls were found. Thus, narcolepsy is unlikely to be a risk factor per se for impaired glucose tolerance or diabetes.

Sleep propensity at daytime as assessed by Multiple Sleep Latency Tests (MSLT) in patients with schizophrenia increases with clozapine and olanzapine.

Sleep propensity at daytime has not been investigated in untreated patients with schizophrenia. Furthermore, while the antipsychotics clozapine and olanzapine are considered to frequently cause 'sleepiness' or 'sedation', this has not been objectified yet. Therefore, 30 patients with schizophrenia were included in this randomized, double-blind study. Sleep propensity was assessed before and after 2, 4 and 6 weeks of treatment with either clozapine or olanzapine using a Multiple Sleep Latency Test (MSLT); in the MSLT, sleep latencies of 5 nap opportunities of 20 min during daytime are averaged. In addition, the number of sleep onsets was recorded. Mean sleep latency in untreated schizophrenic patients was 16.2 ± 0.8 min at baseline. Both antipsychotics induced an increase of sleep propensity as indicated by a shortened sleep latency and more sleep onsets during the treatment period as compared to baseline. These effects were strongest in the morning. Four patients receiving clozapine and 3 patients receiving olanzapine reported subjective sleepiness, in all but one commencing in the first treatment week and persisting until study end. While the mean sleep latency during treatment was significantly shorter in these patients (12.3 ± 0.8 min) than in those without subjective sleepiness (14.9 ± 0.7 min), a short sleep latency was not necessarily associated with subjective sleepiness. In conclusion, mean sleep latency was >36% longer (i.e. sleep propensity was lower) in untreated patients with schizophrenia than in healthy subjects previously consistently reported. Furthermore, clozapine and olanzapine increased sleep propensity in schizophrenic patients. A minority of patients reported subjective sleepiness.