Sleep deprivation and brain energy metabolism : in vivo studies in rats and humans

Sleep deprivation leads to increased subsequent sleep length and depth and to deficits in cognitive performance in humans. In animals extreme sleep deprivation is eventually fatal. The cellular and molecular mechanisms causing the symptoms of sleep deprivation are unclear. This thesis was inspired by the hypothesis that during wakefulness brain energy stores would be depleted, and they would be replenished during sleep. The aim of this thesis was to elucidate the energy metabolic processes taking place in the brain during sleep deprivation. Endogenous brain energy metabolite levels were assessed in vivo in rats and in humans in four separate studies (Studies I-IV). In the first part (Study I) the effects of local energy depletion on brain energy metabolism and sleep were studied in rats with the use of in vivo microdialysis combined with high performance liquid chromatography. Energy depletion induced by 2,4-dinitrophenol infusion into the basal forebrain was comparable to the effects of sleep deprivation: both increased extracellular concentrations of adenosine, lactate, and pyruvate, and elevated subsequent sleep. This result supports the hypothesis of a connection between brain energy metabolism and sleep. The second part involved healthy human subjects (Studies II-IV). Study II aimed to assess the feasibility of applying proton magnetic resonance spectroscopy (1H MRS) to study brain lactate levels during cognitive stimulation. Cognitive stimulation induced an increase in lactate levels in the left inferior frontal gyrus, showing that metabolic imaging of neuronal activity related to cognition is possible with 1H MRS. Study III examined the effects of sleep deprivation and aging on the brain lactate response to cognitive stimulation. No physiologic, cognitive stimulation-induced lactate response appeared in the sleep-deprived and in the aging subjects, which can be interpreted as a sign of malfunctioning of brain energy metabolism. This malfunctioning may contribute to the functional impairment of the frontal cortex both during aging and sleep deprivation. Finally (Study IV), 1H MRS major metabolite levels in the occipital cortex were assessed during sleep deprivation and during photic stimulation. N-acetyl-aspartate (NAA/H2O) decreased during sleep deprivation, supporting the hypothesis of sleep deprivation-induced disturbance in brain energy metabolism. Choline containing compounds (Cho/H2O) decreased during sleep deprivation and recovered to alert levels during photic stimulation, pointing towards changes in membrane metabolism, and giving support to earlier observations of altered brain response to stimulation during sleep deprivation. Based on these findings, it can be concluded that sleep deprivation alters brain energy metabolism. However, the effects of sleep deprivation on brain energy metabolism may vary from one brain area to another. Although an effect of sleep deprivation might not in all cases be detectable in the non-stimulated baseline state, a challenge imposed by cognitive or photic stimulation can reveal significant changes. It can be hypothesized that brain energy metabolism during sleep deprivation is more vulnerable than in the alert state. Changes in brain energy metabolism may participate in the homeostatic regulation of sleep and contribute to the deficits in cognitive performance during sleep deprivation.

Molecular Pathways of Disturbed Sleep and Depression: Studies on Adenosine and Gene Expression Patterns

Background: Adenosine is a potent sleep-promoting substance, and one of its targets is the basal forebrain. Fairly little is known about its mechanism of action in the basal forebrain and about the receptor subtype mediating its regulating effects on sleep homeostasis. Homeostatic deficiency might be one of the causes of the profoundly disturbed sleep pattern in major depressive disorder, which could explain the reduced amounts of delta-activity-rich stages 3 and 4. Since major depression has a relatively high heritability, and on the other hand adenosine regulates sleep homeostasis and might also be involved in mood modulation, adenosine-related genes should be considered for their possible contribution to a predisposition for depression and disturbed sleep in humans. Depression is a complex disorder likely involving the abnormal functioning of several genes. Novel target genes which could serve as the possible common substrates for depression and comorbid disturbed sleep should be identified. In this way specific brain areas related to sleep regulation should be studied by using animal model of depression which represents more homogenous phenotype as compared to humans. It is also important to study these brain areas during the development of depressive-like features to understand how early changes could facilitate pathophysiological changes in depression. Aims and methods: We aimed to find out whether, in the basal forebrain, adenosine induces recovery non-rapid eye movement (NREM) sleep after prolonged waking through the A1 or/and A2A receptor subtype. A1 and A2A receptor antagonists were perfused into the rat basal forebrain during 3 h of sleep deprivation, and the amount of NREM sleep and delta power during recovery NREM sleep were analyzed. We then explored whether polymorphisms in genes related to the metabolism, transport and signaling of adenosine could predispose to depression accompanied by signs of disturbed sleep. DNA from 1423 individuals representative of the Finnish population and including controls and cases with depression, depression accompanied by early morning awakenings and depression accompanied by fatigue, was used in the study to investigate the possible association between polymorphisms from adenosine-related genes and cases. Finally to find common molecular substrates of depression and disturbed sleep, gene expression changes were investigated in specific brain areas in the rat clomipramine model of depression. We focused on the basal forebrain of 3-week old clomipramine-treated rats which develop depressive-like symptoms later in adulthood and on the hypothalamus of adult female clomipramine-treated rats. Results: Blocking of the A1 receptor during sleep deprivation resulted in a reduction of the recovery NREM sleep amount and delta power, whereas A2A receptor antagonism had no effect. Polymorphisms in adenosine-related genes SLC29A3 (equilibrative nucleoside transporter type 3) in women and SLC28A1 (concentrative nucleoside transporter type 1) in men associated with depression alone as well as when accompanied by early morning awakenings and fatigue. In Study III the basal forebrain of postnatal rats treated with clomipramine displayed disturbances in gamma-aminobutyric acid (GABA) receptor type A signaling, in synaptic transmission and possible epigenetic changes. CREB1 was identified as a common transcription denominator which also mediates epigenetic regulation. In the hypothalamus the major changes included the expression of genes in GABA-A receptor pathway, K+ channel-related, glutamatergic and mitochondrial genes, as well as an overexpression of genes related to RNA and mRNA processing. Conclusions: Adenosine plays an important role in sleep homeostasis by promoting recovery NREM sleep via the A1 receptor subtype in the basal forebrain. Also adenosine levels might contribute to the risk of depression with disturbed sleep, since the genes encoding nucleoside transporters showed the strongest associations with depression alone and when accompanied by signs of disturbed sleep in both women and men. Sleep and mood abnormalities in major depressive disorder could be a consequence of multiple changes at the transcriptional level, GABA-A receptor signaling and synaptic transmission in sleep-related basal forebrain and the hypothalamus.

Cellular Mechanisms of Sleep Homeostasis : Studies on Brain Energy Metabolism and Aging

Sleep is governed by a homeostatic process in which the duration and quality of previous wake regulate the subsequent sleep. Active wakefulness is characterized with high frequency cortical oscillations and depends on stimulating influence of the arousal systems, such as the cholinergic basal forebrain (BF), while cessation of the activity in the arousal systems is required for slow wave sleep (SWS) to occur. The site-specific accumulation of adenosine (a by-product of ATP breakdown) in the BF during prolonged waking /sleep deprivation (SD) is known to induce sleep, thus coupling energy demand to sleep promotion. The adenosine release in the BF is accompanied with increases in extracellular lactate and nitric oxide (NO) levels. This thesis was aimed at further understanding the cellular processes by which the BF is involved in sleep-wake regulation and how these processes are affected by aging. The BF function was studied simultaneously at three levels of organization: 1) locally at a cellular level by measuring energy metabolites 2) globally at a cortical level (the out-put area of the BF) by measuring EEG oscillations and 3) at a behavioral level by studying changes in vigilance states. Study I showed that wake-promoting BF activation, particularly with glutamate receptor agonist N-methyl-D-aspatate (NMDA), increased extracellular adenosine and lactate levels and led to a homeostatic increase in the subsequent sleep. Blocking NMDA activation during SD reduced the high frequency (HF) EEG theta (7-9 Hz) power and attenuated the subsequent sleep. In aging, activation of the BF during SD or experimentally with NMDA (studies III, IV), did not induce lactate or adenosine release and the increases in the HF EEG theta power during SD and SWS during the subsequent sleep were attenuated as compared to the young. These findings implicate that increased or continuous BF activity is important for active wake maintenance during SD as well as for the generation of homeostatic sleep pressure, and that in aging these mechanisms are impaired. Study II found that induction of the inducible NO synthase (iNOS) during SD is accompanied with activation of the AMP-activated protein kinase (AMPK) in the BF. Because decreased cellular energy charge is the most common cause for AMPK activation, this finding implicates that the BF is selectively sensitive to the metabolic demands of SD as increases were not found in the cortex. In aging (study III), iNOS expression and extracellular levels of NO and adenosine were not significantly increased during SD in the BF. Furthermore, infusion of NO donor into the BF did not lead to sleep promotion as it did in the young. These findings indicated that the NO (and adenosine) mediated sleep induction is impaired in aging and that it could at least partly be due to the reduced sensitivity of the BF to sleep-inducing factors. Taken together, these findings show that reduced sleep promotion by the BF contributes to the attenuated homeostatic sleep response in aging.

Circadian Rhythm Disruptions and Health

The doctoral thesis defined connections between circadian rhythm disruptions and health problems. Sleep debt, jet-lag, shift work, as well as transitions into and out of the daylight saving time may lead to circadian rhythm disruptions. Disturbed circadian rhythm causes sleep deprivation and decrease of mood and these effects may lead to higher accident rates and trigger mental illnesses. Circadian clock genes are involved in the regulation of the cell cycle and metabolism and thus unstable circadian rhythmicity may also lead to cancer development. In publications I-III it was explored how transitions into and out of the daylight saving time impact the sleep efficiency and the rest-activity cycles of healthy individuals. Also it was explored whether the effect of transition is different in fall as compared to spring, and whether there are subgroup specific differences in the adjustment to transitions into and out of daylight saving time. The healthy participants of studies I-III used actigraphs before and after the transitions and filled in the morningness-eveningness and seasonal pattern assessment questionnaires. In publication IV the incidence of hospital-treated accidents and manic episodes was explored two weeks before and two weeks after the transitions into and out of the daylight saving time in years 1987-2003. In publication V the relationship between circadian rhythm disruption and the prevalence of Non-Hodgkin lymphoma was studied. The study V consisted of all working aged Finns who participated in the national population census in 1970. For our study, all the cancers diagnosed during the years 1971-1995 were extracted from the Finnish Cancer Register and linked with the 1970 census files. In studies I-III it was noticed that transitions into and out of the daylight saving time disturbs the sleep-wake cycle and the sleep efficiency of the healthy participants. We also noticed that short sleepers were more sensitive than long sleepers for sudden changes in the circadian rhythm. Our results also indicated that adaptation to changes in the circadian rhythm is potentially sex, age and chronotype-specific. In study IV no significant increase in the occurrence of hospital treated accidents or manic episodes was noticed. However, interesting observations about the seasonal fluctuation of the occurrence rates of accidents and manic episodes were made. Study V revealed that there might be close relationship between circadian rhythm disruption and cancer. The prevalence of Non-Hodgkin lymphoma was the highest among night workers. The five publications included in this thesis together point out that disturbed circadian rhythms may have adverse effect on health. Disturbed circadian rhythms decrease the quality of sleep and weaken the sleep-wake cycle. A continuous circadian rhythm disruption may also predispose individuals to cancer development. Since circadian rhythm disruptions are common in modern society they might have a remarkable impact on the public health. Thus it is important to continue circadian rhythm research so that better prevention and treatment methods can be developed. Keywords: Circadian rhythm, daylight saving time, manic episodes, accidents, Non-Hodgkin lymphoma 11

Effect of colostrum feeding method and presence of dam on the sleep, rest and sucking behaviour of newborn calves

"In rats, sucking milk reduces anxiety and promotes non-rapid eye movement (NREM) sleep, and in calves it induces resting but the effect on sleep is unknown. Here, we investigated how calves' sleep was affected by colostrum feeding methods. Forty-one calves were blocked by birth date and randomly allotted within blocks to the experimental treatments. Calves were housed for four days either with their dam (DAM) or individually with warm colostrum feeding (2 L four times a day) from either a teat bucket (TEAT) or an open bucket (BUCKET). DAM calves suckled their dam freely. Calves' sleeping and sucking behaviour was filmed continuously for 48 h at the ages of two and three days. Behavioural sleep (BS) was defined as calves resting at least 30 s with their head still and raised (non-rapid eye movement) or with their head against their body or the ground (rapid eye movement, REM). Latency from the end of colostrum feeding to the start of BS was recorded. We compared behaviour of TEAT calves with that of DAM and BUCKET calves using mixed models. Milk meal duration was significantly longer for TEAT calves than for BUCKET calves (mean +/- S.E.M.; 8.3 +/- 0.6 min vs. 5.2 +/- 0.6 min), but equal to that of DAM calves. We found no effect of feeding method on the duration of daily BS (12 h 59 min I h 38 min) but we found a tendency for the daily amount of NREM sleep; BUCKET calves had less NREM sleep per day than TEAT calves (6 h 18 min vs. 7 h 48 min, S.E.M. = 45 min) and also longer latencies from milk ingestion to BS (21.9 +/- 2.0 min vs. 16.2 +/- 2.0 min). DAM calves slept longer bouts than TEAT calves (10.8 +/- 1.0 min vs. 8.3 +/- 1.0 min) and less often (78 +/- 4 vs. 92 +/- 4). Sucking colostrum from a teat bucket compared with drinking from an open"

Social and material deprivation among youth in Finland : causes, consequences, and coping.

Background: Social and material deprivation is associated with poor health, decreased subjective well-being, and limited opportunities for personal development. To date, little is known about the lived experiences of Finnish low-income youths and the general purpose of this study is to fill this gap. Despite the extensive research on socioeconomic income disparities, only a few scholars have addressed the question of how low socioeconomic position is experienced by disadvantaged people themselves. Little is known about the everyday social processes that lead to decreased well-being of economically and socially disadvantaged citizens. Data: The study is based on the data of 65 autobiographical essays written by Finnish low-income youths aged 14-29 (M=23.51, SD=3.95). The research data were originally collected in a Finnish nationwide writing contest “Arkipäivän kokemuksia köyhyydestä” [Everyday Experiences of Poverty] between June and September of 2006. The contest was partaken by 850 Finnish writers. Methods and key concepts: Autobiographical narratives (N=65) of low-income youths were analyzed based on grounded theory methodology (GTM). The analysis was not built on specific pre-conceived categorizations; it was guided by the paradigm model and so-called “sensitizing concepts”. The concepts this study utilized were based on the research literature on socioeconomic inequalities, resilience, and coping. Socioeconomic inequalities refer to unequal distribution of resources, such as income, social status, and health, between social groups. The concept of resilience refers to an individual’s capacity to cope despite existing risk factors and conditions that are harmful to health and well-being. Coping strategies can be understood as ways by which a person tries to cope with psychological stress in a situation where internal or externals demands exceed one’s resources. The ways to cope are cognitive or behavioral efforts by which individual tries to relieve the stress and gain new resources. Lack of material and social resources is associated with increased exposure to health-related stressors during the life-course. Aims: The first aim of this study is to illustrate how youths with low socioeconomic status perceive the causes and consequences of their social and material deprivation. The second aim is to describe what kind of coping strategies youths employ to cope in their everyday life. The third aim is to build an integrative conceptual framework based on the relationships between causes, consequences, and individual coping strategies associated with deprivation. The analysis was carried out through systematic coding and orderly treatment of the data based on the grounded theory methodology. Results: Finnish low-income youths attributed the primary causes of deprivation to their family background, current socioeconomic status, sudden life changes, and contextual factors. Material and social deprivation was associated with various kinds of negative psychological, social, and material consequences. Youths used a variety of coping strategies that were identified as psychological, social, material, and functional-behavioral. Finally, a conceptual framework was formulated to link the findings together. In the discussion, the results were compared and contrasted to the existing research literature. The main references of the study were: Coping: Aldwin (2007); Lazarus & Folkman (1984); Hobfoll (1989, 2001, 2002). Deprivation: Larivaara, Isola, & Mikkonen (2007); Lister (2004); Townsend (1987); Raphael (2007). Health inequalities: Dahlgren & Whitehead (2007); Lynch. et al. (2000); Marmot & Wilkinson (2006); WHO (2008). Methods: Charmaz (2006); Flick (2009); Strauss & Corbin (1990). Resilience: Cutuli & Masten (2009); Luthar (2006).

Novel Mass Spectrometric Analysis Methods for Anabolic Androgenic Steroids in Sports Drug Testing

The feasibility of different modern analytical techniques for the mass spectrometric detection of anabolic androgenic steroids (AAS) in human urine was examined in order to enhance the prevalent analytics and to find reasonable strategies for effective sports drug testing. A comparative study of the sensitivity and specificity between gas chromatography (GC) combined with low (LRMS) and high resolution mass spectrometry (HRMS) in screening of AAS was carried out with four metabolites of methandienone. Measurements were done in selected ion monitoring mode with HRMS using a mass resolution of 5000. With HRMS the detection limits were considerably lower than with LRMS, enabling detection of steroids at low 0.2-0.5 ng/ml levels. However, also with HRMS, the biological background hampered the detection of some steroids. The applicability of liquid-phase microextraction (LPME) was studied with metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol. Factors affecting the extraction process were studied and a novel LPME method with in-fiber silylation was developed and validated for GC/MS analysis of the danazol metabolite. The method allowed precise, selective and sensitive analysis of the metabolite and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine without any other steps in sample preparation. Liquid chromatographic/tandem mass spectrometric (LC/MS/MS) methods utilizing electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were developed and applied for detection of oxandrolone and metabolites of stanozolol and 4-chlorodehydromethyltestosterone in urine. All methods exhibited high sensitivity and specificity. ESI showed, however, the best applicability, and a LC/ESI-MS/MS method for routine screening of nine 17-alkyl-substituted AAS was thus developed enabling fast and precise measurement of all analytes with detection limits below 2 ng/ml. The potential of chemometrics to resolve complex GC/MS data was demonstrated with samples prepared for AAS screening. Acquired full scan spectral data (m/z 40-700) were processed by the OSCAR algorithm (Optimization by Stepwise Constraints of Alternating Regression). The deconvolution process was able to dig out from a GC/MS run more than the double number of components as compared with the number of visible chromatographic peaks. Severely overlapping components, as well as components hidden in the chromatographic background could be isolated successfully. All studied techniques proved to be useful analytical tools to improve detection of AAS in urine. Superiority of different procedures is, however, compound-dependent and different techniques complement each other.

Univariate, bivariate, and multivariate methods in corpus-based lexicography : A study of synonymy

In this dissertation, I present an overall methodological framework for studying linguistic alternations, focusing specifically on lexical variation in denoting a single meaning, that is, synonymy. As the practical example, I employ the synonymous set of the four most common Finnish verbs denoting THINK, namely ajatella, miettiä, pohtia and harkita ‘think, reflect, ponder, consider’. As a continuation to previous work, I describe in considerable detail the extension of statistical methods from dichotomous linguistic settings (e.g., Gries 2003; Bresnan et al. 2007) to polytomous ones, that is, concerning more than two possible alternative outcomes. The applied statistical methods are arranged into a succession of stages with increasing complexity, proceeding from univariate via bivariate to multivariate techniques in the end. As the central multivariate method, I argue for the use of polytomous logistic regression and demonstrate its practical implementation to the studied phenomenon, thus extending the work by Bresnan et al. (2007), who applied simple (binary) logistic regression to a dichotomous structural alternation in English. The results of the various statistical analyses confirm that a wide range of contextual features across different categories are indeed associated with the use and selection of the selected think lexemes; however, a substantial part of these features are not exemplified in current Finnish lexicographical descriptions. The multivariate analysis results indicate that the semantic classifications of syntactic argument types are on the average the most distinctive feature category, followed by overall semantic characterizations of the verb chains, and then syntactic argument types alone, with morphological features pertaining to the verb chain and extra-linguistic features relegated to the last position. In terms of overall performance of the multivariate analysis and modeling, the prediction accuracy seems to reach a ceiling at a Recall rate of roughly two-thirds of the sentences in the research corpus. The analysis of these results suggests a limit to what can be explained and determined within the immediate sentential context and applying the conventional descriptive and analytical apparatus based on currently available linguistic theories and models. The results also support Bresnan’s (2007) and others’ (e.g., Bod et al. 2003) probabilistic view of the relationship between linguistic usage and the underlying linguistic system, in which only a minority of linguistic choices are categorical, given the known context – represented as a feature cluster – that can be analytically grasped and identified. Instead, most contexts exhibit degrees of variation as to their outcomes, resulting in proportionate choices over longer stretches of usage in texts or speech.

Genotyping for genetic association studies: Methods and applications

In this thesis, two separate single nucleotide polymorphism (SNP) genotyping techniques were set up at the Finnish Genome Center, pooled genotyping was evaluated as a screening method for large-scale association studies, and finally, the former approaches were used to identify genetic factors predisposing to two distinct complex diseases by utilizing large epidemiological cohorts and also taking environmental factors into account. The first genotyping platform was based on traditional but improved restriction-fragment-length-polymorphism (RFLP) utilizing 384-microtiter well plates, multiplexing, small reaction volumes (5 µl), and automated genotype calling. We participated in the development of the second genotyping method, based on single nucleotide primer extension (SNuPeTM by Amersham Biosciences), by carrying out the alpha- and beta tests for the chemistry and the allele-calling software. Both techniques proved to be accurate, reliable, and suitable for projects with thousands of samples and tens of markers. Pooled genotyping (genotyping of pooled instead of individual DNA samples) was evaluated with Sequenom s MassArray MALDI-TOF, in addition to SNuPeTM and PCR-RFLP techniques. We used MassArray mainly as a point of comparison, because it is known to be well suited for pooled genotyping. All three methods were shown to be accurate, the standard deviations between measurements being 0.017 for the MassArray, 0.022 for the PCR-RFLP, and 0.026 for the SNuPeTM. The largest source of error in the process of pooled genotyping was shown to be the volumetric error, i.e., the preparation of pools. We also demonstrated that it would have been possible to narrow down the genetic locus underlying congenital chloride diarrhea (CLD), an autosomal recessive disorder, by using the pooling technique instead of genotyping individual samples. Although the approach seems to be well suited for traditional case-control studies, it is difficult to apply if any kind of stratification based on environmental factors is needed. Therefore we chose to continue with individual genotyping in the following association studies. Samples in the two separate large epidemiological cohorts were genotyped with the PCR-RFLP and SNuPeTM techniques. The first of these association studies concerned various pregnancy complications among 100,000 consecutive pregnancies in Finland, of which we genotyped 2292 patients and controls, in addition to a population sample of 644 blood donors, with 7 polymorphisms in the potentially thrombotic genes. In this thesis, the analysis of a sub-study of pregnancy-related venous thromboses was included. We showed that the impact of factor V Leiden polymorphism on pregnancy-related venous thrombosis, but not the other tested polymorphisms, was fairly large (odds ratio 11.6; 95% CI 3.6-33.6), and increased multiplicatively when combined with other risk factors such as obesity or advanced age. Owing to our study design, we were also able to estimate the risks at the population level. The second epidemiological cohort was the Helsinki Birth Cohort of men and women who were born during 1924-1933 in Helsinki. The aim was to identify genetic factors that might modify the well known link between small birth size and adult metabolic diseases, such as type 2 diabetes and impaired glucose tolerance. Among ~500 individuals with detailed birth measurements and current metabolic profile, we found that an insertion/deletion polymorphism of the angiotensin converting enzyme (ACE) gene was associated with the duration of gestation, and weight and length at birth. Interestingly, the ACE insertion allele was also associated with higher indices of insulin secretion (p=0.0004) in adult life, but only among individuals who were born small (those among the lowest third of birth weight). Likewise, low birth weight was associated with higher indices of insulin secretion (p=0.003), but only among carriers of the ACE insertion allele. The association with birth measurements was also found with a common haplotype of the glucocorticoid receptor (GR) gene. Furthermore, the association between short length at birth and adult impaired glucose tolerance was confined to carriers of this haplotype (p=0.007). These associations exemplify the interaction between environmental factors and genotype, which, possibly due to altered gene expression, predisposes to complex metabolic diseases. Indeed, we showed that the common GR gene haplotype associated with reduced mRNA expression in thymus of three individuals (p=0.0002).