Intracerebral Adenosine During Sleep Deprivation: A Meta-Analysis and New Experimental Data.

The neuroregulator adenosine is involved in sleep-wake control. Basal forebrain (BF) adenosine levels increase during sleep deprivation. Only a few studies have addressed the effect of sleep deprivation on extracellular adenosine concentrations in other brain regions. In this paper, we describe a microdialysis experiment as well as a meta-analysis of published data. The 64 h microdialysis experiment determined the extracellular adenosine and adenosine monophosphate (AMP) concentrations in the medial prefrontal cortex of rats before, during and after 12 h of sleep deprivation by forced locomotion. The meta-analysis comprised published sleep deprivation animal experiments measuring adenosine by means of microdialysis. In the animal experiment, the overall median adenosine concentration was 0.36 nM and ranged from 0.004 nM to 27 nM. No significant differences were observed between the five conditions: 12 h of wash-out, baseline light phase, baseline dark phase, 12 h of sleep deprivation and 12 h of subsequent recovery. The overall median AMP concentration was 0.10 nM and ranged from 0.001 nM to 7.56 nM. Median AMP concentration increased during sleep deprivation (T = 47; p = 0.047) but normalised during subsequent recovery. The meta-analysis indicates that BF dialysate adenosine concentrations increase with 74.7% (95% CI: 54.1-95.3%) over baseline during sleep deprivation. Cortex dialysate adenosine concentrations during sleep deprivation were so far only reported by 2 publications. The increase in adenosine during sleep deprivation might be specific to the BF. At this stage, the evidence for adenosine levels in other brain regions is based on single experiments and insufficient for generalised conclusions. Further experiments are currently still warranted.

Intracerebral microdialysis of adenosine and adenosine monophosphate - a systematic review and meta-regression analysis of baseline concentrations.

Microdialysis is a method to study the extracellular space in vivo, based on the principle of diffusion. It can be used to measure various small molecules including the neuroregulator adenosine. Baseline levels of the compounds measured with microdialysis vary over studies. We systematically reviewed the literature to investigate the full range of reported baseline concentrations of adenosine and adenosine monophosphate in microdialysates. We performed a meta-regression analysis to study the influence of flow rate, probe membrane surface area, species, brain area and anaesthesia versus freely behaving, on the adenosine concentration. Baseline adenosine concentrations in microdialysates ranged from 0.8 to 2100 nM. There was limited evidence on baseline adenosine monophosphate concentrations in microdialysates. Across studies, we found effects of flow rate and anaesthesia versus freely behaving on dialysate adenosine concentrations (p ≤ 0.001), but not of probe membrane surface, species, or brain area (p ≥ 0.14). With increasing flow rate, adenosine concentrations decreased. With anaesthesia, adenosine concentrations increased. The effect of other predictor variables on baseline adenosine concentrations, for example, post-surgical recovery time, could not be analysed because of a lack of reported data. This study shows that meta-regression can be used as an alternative to new animal experiments to answer research questions in the field of neurochemistry. However, current levels of reporting of primary studies are insufficient to reach the full potential of this approach; 63 out of 133 studies could not be included in the analysis because of insufficient reporting, and several potentially relevant factors had to be excluded from the analyses. The level of reporting of experimental detail needs to improve.

Repeated psychosocial stress at night, but not day, affects the central molecular clock.

We have recently demonstrated that the outcome of repeated social defeat (SD) on behavior, physiology and immunology is more negative when applied during the dark/active phase as compared with the light/inactive phase of male C57BL/6 mice. Here, we investigated the effects of the same stress paradigm, which combines a psychosocial and novelty stressor, on the circadian clock in transgenic PERIOD2::LUCIFERASE (PER2::LUC) and wildtype (WT) mice by subjecting them to repeated SD, either in the early light phase (social defeat light = SDL) or in the early dark phase (social defeat dark = SDD) across 19 days. The PER2::LUC rhythms and clock gene mRNA expression were analyzed in the suprachiasmatic nucleus (SCN) and the adrenal gland, and PER2 protein expression in the SCN was assessed. SDD mice showed increased PER2::LUC rhythm amplitude in the SCN, reduced Per2 and Cryptochrome1 mRNA expression in the adrenal gland, and increased PER2 protein expression in the posterior part of the SCN compared with single-housed control (SHC) and SDL mice. In contrast, PER2::LUC rhythms in the SCN of SDL mice were not affected. However, SDL mice exhibited a 2-hour phase advance of the PER2::LUC rhythm in the adrenal gland compared to SHC mice. Furthermore, plasma levels of brain-derived neurotrophic factor (BDNF) and BDNF mRNA in the SCN were elevated in SDL mice. Taken together, these results show that the SCN molecular rhythmicity is affected by repeated SDD, but not SDL, while the adrenal peripheral clock is influenced mainly by SDL. The observed increase in BDNF in the SDL group may act to protect against the negative consequences of repeated psychosocial stress.

TrkB-mediated protection against circadian sensitivity to noise trauma in the murine cochlea.

Noise-induced hearing loss (NIHL) is a debilitating sensory impairment affecting 10%-15% of the population, caused primarily through damage to the sensory hair cells or to the auditory neurons. Once lost, these never regenerate [1], and no effective drugs are available [2, 3]. Emerging evidence points toward an important contribution of synaptic ribbons in the long-term coupling of the inner hair cell and afferent neuron synapse to maintain hearing [4]. Here we show in nocturnal mice that night noise overexposure triggers permanent hearing loss, whereas mice overexposed during the day recover to normal auditory thresholds. In view of this time-dependent sensitivity, we identified a self-sustained circadian rhythm in the isolated cochlea, as evidenced by circadian expression of clock genes and ample PERIOD2::LUCIFERASE oscillations, originating mainly from the primary auditory neurons and hair cells. The transcripts of the otoprotecting brain-derived neurotrophic factor (BDNF) showed higher levels in response to day noise versus night noise, suggesting that BDNF-mediated signaling regulates noise sensitivity throughout the day. Administration of a selective BDNF receptor, tropomyosin-related kinase type B (TrkB), in the night protected the inner hair cell's synaptic ribbons and subsequent full recovery of hearing thresholds after night noise overexposure. The TrkB agonist shifted the phase and boosted the amplitude of circadian rhythms in the isolated cochlea. These findings highlight the coupling of circadian rhythmicity and the TrkB receptor for the successful prevention and treatment of NIHL.

Inter-tissue networks between the basal forebrain, hippocampus, and prefrontal cortex in a model for depression caused by disturbed sleep.

Disturbances in sleep are encountered in the majority of patients with depressive disorder. To elucidate the molecular mechanisms behind this relationship, we examined gene expression changes in a rodent model for disturbed sleep and depression. The animals were treated with daily injections of clomipramine to affect their sleep during early infancy. This early interference with sleep is known to induce depression-like behavior in adult animals. After 2 weeks of treatment, the change in gene expression was examined using the Affymetrix Rat 230.2 chip. We studied the gene expression in the basal forebrain, hippocampus, and frontal cortex and combined the results to reveal the otherwise indissectible networks between and around the tissues. The major disrupted pathways between the three brain areas were related to synaptic transmission, regulation of translation, and ubiquitinylation. The involved pathways were within the cellular components of the axons, growth cones, melanosomes, and pigment granules. A network analysis allowing for additional interactors, in the form of chemicals or gene products, revealed a disturbed communicational network between the different brain areas. This disturbed network is centered around serotonin, Mn(II), and Rhoa. The findings elucidate inter-tissue pathways and networks in the brain that are involved in sleep and mood regulation. The findings are of uttermost interest, some are quite predictable and obvious, but some are novel or have only been proposed by rare theoretical speculations (such as the melanosome and Mn(II) involvement). Equally important as the findings are the methods described in this article. In this study, we present two novel simple ways to perform system biological analysis based on gene expression array data. We used two already existing tools in a new way, and by careful planning of the input data, managed to extrapolate intricate hidden inter-tissue networks to build a molecular picture of disease.

Slice preparation, organotypic tissue culturing and luciferase recording of clock gene activity in the suprachiasmatic nucleus.

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function. The SCN is a small, well-defined bilateral structure located right above the optic chiasm. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 µm (length, rostrocaudal axis) x 424 µm (width) x 390 µm (height). To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane, a technique developed for SCN tissue culture by Yamazaki et al. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi.