Miroslaw Mackiewicz

Modulation of IL-1β gene expression in the rat CNS during sleep deprivation

We hypothesize that sleep homeostasis involves, at least in part, the immune system modulator interleukin-1β (IL-1β). Using the reverse transcription-polymerase chain reaction, IL-1β mRNA levels in the rat CNS were evaluated after a period of sleep deprivation. In addition, IL-1β gene expression was analyzed before the projected onset of activity and rest phase in free-running animals. No changes in IL-1β mRNA were observed in the circadian cycle, but 24 h of sleep deprivation resulted in a 2-fold increase in the level of IL-1β mRNA in the hypothalamus and in the brain stem compared with controls (p< 0.0002 and (p < 0.0001 respectively). The alteration in IL-1β mRNA levels following sleep deprivation supports the hypothesis that modulation of IL-1β gene expression is involved in the sleep homeostatic process.

Molecular Mechanisms of Sleep and Wakefulness

Major questions on the biology of sleep include the following: what are the molecular functions of sleep; why can wakefulness only be sustained for defined periods before there is behavioral impairment; what genes contribute to the individual differences in sleep and the response to sleep deprivation? Behavioral criteria to define sleep have facilitated identification of sleep states in a number of different model systems: Drosophila, zebrafish, and Caenorhabditis elegans. Each system has unique strengths. Studies in these model systems are identifying conserved signaling mechanisms regulating sleep that are present in mammals. For example, the PKA‐CREB signaling mechanism promotes wakefulness in Drosophila, mice, and C. elegans. Microarray studies indicate that genes whose expression is upregulated during sleep are involved in macromolecule biosynthesis (proteins, lipids [including cholesterol], heme). Thus, a key function of sleep is likely to be macromolecule synthesis. Moreover, in all species studied to date, there is upregulation of the molecular chaperone BiP with extended wakefulness. Sleep deprivation leads to cellular ER stress in brain and the unfolded protein response. Identification of genes regulating sleep has the potential for translational studies to elucidate the genetics of sleep and response to sleep deprivation in humans.

Serotonin receptor mRNA expression in the hypoglossal motor nucleus.

Brainstem serotonin (5-HT)-containing cells are remarkable for their widespread axonal projections and having their highest activity during wakefulness and lowest during rapid eye movement sleep. One important site of action of 5-HT is on upper airway motoneurons. However, which of the 14 known 5-HT receptors mediate the effects is uncertain. We used the reverse transcriptase/polymerase chain reaction to detect mRNA for six distinct 5-HT receptors (1A, 1B, 2A, 2C, 3 and 7) in 50 nl micro-punches collected from the hypoglossal (XII) motor nucleus and, for comparison, from the viscerosensory nucleus of the solitary tract (NTS) in adult rats. The relative abundance of the distinct mRNAs was characterized by the minimal number of amplification cycles (25-40) necessary to detect a given mRNA. In the XII nucleus, mRNA for type 1B, 2A and 2C receptors was detectable after 29-31 cycles, detection of type 3 and 7 receptor mRNA required 33-35 cycles; and type 1A receptor mRNA was not detected. In the NTS, detection of mRNA for type 1B, 2C and 7 receptors required 31-33 cycles; type 1A receptor mRNA required 39 cycles; and type 2A receptor mRNA was not detected. The data from the XII nucleus demonstrate that not only the previously recognized type 1B, 2A and 2C receptors, but also type 3 and 7 receptors have the potential to mediate serotonergic effects in XII motoneurons.

What are microarrays teaching us about sleep

Many fundamental questions about sleep remain unanswered. The presence of sleep across phyla suggests that it must serve a basic cellular and/or molecular function. Microarray studies, performed in several model systems, have identified classes of genes that are sleep-state regulated. This has led to the following concepts: first, a function of sleep is to maintain synaptic homeostasis; second, sleep is a stage of macromolecule biosynthesis; third, extending wakefulness leads to downregulation of several important metabolic pathways; and, fourth, extending wakefulness leads to endoplasmic reticulum stress. In human studies, microarrays are being applied to the identification of biomarkers for sleepiness and for the common debilitating condition of obstructive sleep apnea.

Enzymes of adenosine metabolism in the brain: diurnal rhythm and the effect of sleep deprivation.

Adenosine plays a role in promoting sleep, an effect that is thought to be mediated in the basal forebrain. Adenosine levels vary in this region with prolonged wakefulness in a unique way. The basis for this is unknown. We examined, in rats, the activity of the major metabolic enzymes for adenosine – adenosine deaminase, adenosine kinase, ecto‐ and cytosolic 5′‐nucleotidase – in sleep/wake regulatory regions as well as cerebral cortex, and how the activity varies across the day and with sleep deprivation. There were robust spatial differences for the activity of adenosine deaminase, adenosine kinase, and cytosolic and ecto‐5′‐nucleotidase. However, the basal forebrain was not different from other sleep/wake regulatory regions apart from the tuberomammillary nucleus. All adenosine metabolic enzymes exhibited diurnal variations in their activity, albeit not in all brain regions. Activity of adenosine deaminase increased during the active period in the ventrolateral pre‐optic area but decreased significantly in the basal forebrain. Enzymatic activity of adenosine kinase and cytosolic‐5′‐nucleotidase was higher during the active period in all brain regions tested. However, the activity of ecto‐5′‐nucleotidase was augmented during the active period only in the cerebral cortex. This diurnal variation may play a role in the regulation of adenosine in relationship to sleep and wakefulness across the day. In contrast, we found no changes specifically with sleep deprivation in the activity of any enzyme in any brain region. Thus, changes in adenosine with sleep deprivation are not a consequence of alterations in adenosine enzyme activity.

α1B receptors are the main postsynaptic mediators of adrenergic excitation in brainstem motoneurons, a single-cell RT-PCR study

Norepinephrine (NE) is an important modulator of brainstem motoneurons. It is released at high levels during wakefulness, whereas its reduced release during sleep may contribute to motor suppression, including upper airway hypotonia. To identify the receptors that mediate postsynaptic effects of NE in brainstem motoneurons of juvenile and adult rats, we determined the pattern of adrenoceptor mRNA expression and co-expression in retrogradely labeled and acutely dissociated hypoglossal (XII) motoneurons (n=121) using single-cell, real-time reverse transcription-polymerase chain reaction (RT-PCR). The alpha(1B) receptor mRNA was present in most motoneurons (33/39 or 85%). The remaining six adrenoceptor mRNA species investigated were consistently present in micropunches of tissue extracted from the XII nucleus, but were either rarely expressed in individual motoneurons (alpha(1A) mRNA in 15%, alpha(1D) in 14%, alpha(2B/C) in 2% of cells) or absent (alpha(2A), beta(1) and beta(2)). When present, the alpha(1A) and alpha(1D) mRNAs were co-expressed with alpha(1B) mRNA. The adrenoceptor mRNA expression profiles in dissociated locus coeruleus and inferior olive neurons were significantly different. We conclude that postsynaptic effects of NE in XII motoneurons are primarily mediated by alpha(1B) receptors; the effects ascribed to alpha(2) and/or beta adrenoceptors may be exerted presynaptically.

Analysis of the QTL for sleep homeostasis in mice: Homer1a is a likely candidate

Electroencephalographic oscillations in the frequency range of 0.5-4 Hz, characteristic of slow-wave sleep (SWS), are often referred to as the delta oscillation or delta power. Delta power reflects sleep intensity and correlates with the homeostatic response to sleep loss. A published survey of inbred strains of mice demonstrated that the time course of accumulation of delta power varied among inbred strains, and the segregation of the rebound of delta power in BxD recombinant inbred strains identified a genomic region on chromosome 13 referred to as the delta power in SWS (or Dps1). The quantitative trait locus (QTL) contains genes that modify the accumulation of delta power after sleep deprivation. Here, we narrow the QTL using interval-specific haplotype analysis and present a comprehensive annotation of the remaining genes in the Dps1 region with sequence comparisons to identify polymorphisms within the coding and regulatory regions. We established the expression pattern of selected genes located in the Dps1 interval in sleep and wakefulness in B6 and D2 parental strains. Taken together, these steps reduced the number of potential candidate genes that may underlie the accumulation of delta power after sleep deprivation and explain the Dps1 QTL. The strongest candidate gene is Homer1a, which is supported by expression differences between sleep and wakefulness and the SNP polymorphism in the upstream regulatory regions.

Glycogen in the brain of Drosophila melanogaster: diurnal rhythm and the effect of rest deprivation.

One function of sleep is thought to be the restoration of energy stores in the brain depleted during wakefulness. One such energy store found in mammalian brains is glycogen. Many of the genes involved in glycogen regulation in mammals have also been found in Drosophila melanogaster and rest behavior in Drosophila has recently been shown to have the characteristics of sleep. We therefore examined, in the fly, variation in the glycogen contents of the brain, the whole head and the body throughout the rest/activity cycle and after rest deprivation. Glycogen in the brain varies significantly throughout the day (p = 0.001) and is highest during rest and lowest while flies are active. Glycogen levels in the whole head and body do not show diurnal variation. Brain glycogen drops significantly when flies are rest deprived for 3 h (p = 0.034) but no significant differences are observed after 6 h of rest deprivation. In contrast, glycogen is significantly depleted in the body after both 3 and 6 h of rest deprivation (p < 0.0001 and p < 0.0001, respectively). Glycogen in the fly brain changes in relationship to rest and activity and demonstrates a biphasic response to rest deprivation similar to that observed in mammalian astrocytes in culture.

AMP-activated protein kinase phosphorylation in brain is dependent on method of killing and tissue preparation

AMP‐activated protein kinase (AMPK) is activated when the catalytic α subunit is phosphorylated on Thr172 and therefore, phosphorylation of the α subunit is used as a measure of activation. However, measurement of α subunit of AMPK (α‐AMPK) phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring α‐AMPK phosphorylation in the mouse brain, we compared different methods of killing and tissue preparation. We found that freeze/thawing samples after homogenization on ice dramatically increased α‐AMPK phosphorylation in mice killed by cervical dislocation. Killing of mice by focused microwave irradiation, which rapidly heats the brain and causes enzymatic inactivation, prevented the freeze/thaw‐induced increase in α‐AMPK phosphorylation and similar levels of phosphorylation were observed compared with mice killed with cervical dislocation without freeze/thawing of samples. Sonication of samples in hot 1% sodium dodecyl sulfate blocked the freeze/thaw‐induced increase in α‐AMPK phosphorylation, but phosphorylation was higher in mice killed by cervical dislocation compared with mice killed by focused microwave irradiation. These results demonstrate that α‐AMPK phosphorylation is dependent on method of killing and tissue preparation and that α‐AMPK phosphorylation can increase in a manner that does not reflect biological alterations.

Activity of adenosine deaminase in the sleep regulatory areas of the rat CNS

There are data to support the notion that adenosine (ADO), a neuromodulator in the CNS, is an important regulator of sleep homeostasis. It has been demonstrated that ADO agonists and antagonists strongly impact upon sleep. In addition, the level of adenosine varies across the sleep/wake cycle and increases following sleep deprivation. Adenosine deaminase (ADA) is a key enzyme involved in the metabolism of ADO. We questioned, therefore, whether there are differences in adenosine deaminase activity in brain regions relevant to sleep regulation. We found that ADA exhibits a characteristic spatial pattern of activity in the rat CNS with the lowest activity in the parietal cortex and highest in the region of the tuberomammillary nucleus (15.0+/-4.8 and 63.4+/-28.0 nmoles/mg protein/15 min, mean+/-S.D., respectively). There were significant differences among the brain regions by one-way ANOVA (F=31.33, df=6, 123, P=0.0001). The regional differences in ADA activity correlate with variations in the level of its mRNA. This suggests that spatial differences in ADA activity are the result of changes in the expression of the ADA gene. We postulate that adenosine deaminase plays an important role in the mechanism that controls regional concentration of adenosine in the brain and thus, it is a part of the sleep-wake regulatory mechanism.