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The mesolimbic dopamine pathway between the ventral tegmental area (VTA) and the nucleus accumbens (NAc) plays a central role in motivational behaviors. Recent findings indicate that the VTA and NAc are also involved in sleep/wake regulation – the topic of this review. First, we present an overview of the growing evidence from rodent studies revealing a wake-regulatory role of VTA dopamine neurons. We also discuss brain areas and their neurotransmitters or neuromodulators that may regulate the activity of wake-promoting VTA dopamine neurons. This is followed by a summary of current knowledge of the role of the NAc in regulating slow-wave sleep and a discussion of where and how this control of sleep physiology might be regulated by upstream neurotransmitters and neuromodulators, including dopamine and the classic somnogen adenosine. [ABSTRACT FROM AUTHOR]

Elongation Factor-2 Kinase (eEF2K) in an unusual mammalian enzyme that has one known substrate, elongation factor-2. It belongs to a class of kinases, called alpha kinases, that has little sequence identity to the > 500 conventional protein kinases, but performs the same reaction and has similar catalytic residues. The phosphorylation of eEF2 blocks translation elongation, which is thought to be critical to regulating cellular energy usage. Here we report a system for discovering new substrates of alpha kinases and identify the first new substrates of eEF2K including AMPK and alpha4, and determine a sequence motif for the kinase that shows a requirement for threonine residues as the target of phosphorylation. These new substrates suggest that eEF2K has a more diverse role in regulating cellular energy usage that involves multiple pathways and regulatory feedback. [ABSTRACT FROM AUTHOR]

Summary Here, we show neuronal inactivation-induced presynaptic remodeling and involvement of the mammalian homolog of Diaphanous (mDia) and Rho-associated coiled-coil-containing kinase (ROCK), Rho-regulated modulators of actin and myosin, in this process. We find that social isolation induces inactivation of nucleus accumbens (NAc) neurons associated with elevated anxiety-like behavior, and that mDia in NAc neurons is essential in this process. Upon inactivation of cultured neurons, mDia induces circumferential actin filaments around the edge of the synaptic cleft, which contract the presynaptic terminals in a ROCK-dependent manner. Social isolation induces similar mDia-dependent presynaptic contraction at GABAergic synapses from NAc neurons in the ventral tegmental area (VTA) associated with reduced synaptic efficacy. Optogenetic stimulation of NAc neurons rescues the anxiety phenotype, and injection of a specific ROCK inhibitor, Y-27632, into the VTA reverses both presynaptic contraction and the behavioral phenotype. mDia-ROCK signaling thus mediates actin-dependent presynaptic remodeling in inactivated NAc neurons, which underlies synaptic plasticity in emotional behavioral responses. [ABSTRACT FROM AUTHOR]

Energy homeostasis in eukaryotic cells is a complex and fundamental process that is misregulated in several human diseases. A key component of energy regulation is a process called autophagy that involves the recycling of cellular components. There has been much recent interest in studying the mechanism of autophagy to understand an important cellular process and to evaluate the therapeutic potential in targeting autophagy. Activation of a kinase called ULK1 initiates autophagy by driving downstream pathways that lead to the formation of double membrane bound vesicles that surround the cellular contents that are to be degraded. Here, we report the discovery of an inhibitor of ULK1 with improved selectivity and a high-resolution crystal structure of the compound bound to the kinase, which will be useful tools for studying autophagy in cells. [ABSTRACT FROM AUTHOR]

Highlights: [•] Protocols are the foundation of an offset program. [•] Using sample projects, we “road test” landfill, manure and afforestation protocols from 5 programs. [•] For a given project, we find large variation in the volume of offsets generated. [•] Harmonization of protocols can increase the likelihood that “a ton is a ton”. [•] Harmonization can enhance prospects for linking emission trading systems. [ABSTRACT FROM AUTHOR]

Highlights: [•] The dorsal striatum enhances wakefulness and the nucleus accumbens promotes sleep. [•] The external globus pallidus regulates sleep–wake behavior through cortical activation. [•] Adenosine A2A receptors in the nucleus accumbens shell are critical for caffeine arousal. [•] The nucleus accumbens has an intrinsic role in the sleep/wake regulatory network. [•] The motivational state is a fundamental regulator of sleep and wakefulness. [ABSTRACT FROM AUTHOR]

The basal ganglia (BG) are involved in motor function, habit formation, and reward or addictive behaviors, but the question as to how the BG integrate arousal with these fundamental striatal functions has only recently received much attention. Findings based on electrophysiology, neurotoxic lesioning, and the use of transgenic animals have established that the striatum and globus pallidus are key structural elements for the control of sleep and wakefulness. Here, we discuss emerging anatomical and molecular mechanisms of sleep–wake regulation at work in the BG. Furthermore, we propose a model whereby adenosine and dopamine receptors in the nucleus accumbens (NAc) are involved in the integration of behavioral processes and the induction of wakefulness through cortical activation. [ABSTRACT FROM AUTHOR]

Sleep is generally viewed as a period of recovery, but how the supply of cerebral blood flow (CBF) changes across sleep/wake states has remained unclear. Here, we directly observe red blood cells (RBCs) within capillaries, where the actual substance exchange between the blood and neurons/glia occurs, by two-photon microscopy. Across multiple cortical areas, average capillary CBF is largely increased during rapid eye movement (REM) sleep, whereas it does not differ between periods of active wakefulness and non-REM sleep. Capillary RBC flow during REM sleep is further elevated following REM sleep deprivation, suggesting that capillary CBF reflects REM sleep pressure. At the molecular level, signaling via adenosine A2a receptors is crucial; in A2a-KO mice, capillary CBF upsurge during REM sleep is dampened, and effects of REM sleep pressure are abolished. These results provide evidence regarding the dynamics of capillary CBF across sleep/wake states and insights to the underlying mechanisms. [Display omitted] • A method to directly measure capillary CBF across sleep/wake is developed • Capillary CBF is highest during REM sleep • Capillary CBF is comparable between active wakefulness and non-REM sleep • A2aR signaling is crucial for the CBF upsurge during REM sleep Tsai et al. develop a method to directly measure capillary cerebral blood flow (CBF) across the sleep/wake cycle in mice. Capillary CBF is drastically increased during REM sleep, while it is comparable between wakefulness and non-REM sleep. Knocking out the adenosine A2a receptor specifically reduces capillary CBF during REM sleep. [ABSTRACT FROM AUTHOR]

2-Oxoadipate dehydrogenase (E1a, also known as DHTKD1, dehydrogenase E1, and transketolase domain-containing protein 1) is a thiamin diphosphate-dependent enzyme and part of the 2-oxoadipate dehydrogenase complex (OADHc) in L-lysine catabolism. Genetic findings have linked mutations in the DHTKD1 gene to several metabolic disorders. These include -aminoadipic and -ketoadipic aciduria (AMOXAD), a rare disorder of L-lysine, L-hydroxylysine, and L-tryptophan catabolism, associated with clinical presentations such as developmental delay, mild-to-severe intellectual disability, ataxia, epilepsy, and behavioral disorders that cannot currently be managed by available treatments. A heterozygous missense mutation, c.2185G3A(p.G729R), inDHTKD1has been identified in most AMOXAD cases. Here, we report that the G729R E1a variant when assembled into OADHc in vitro displays a 50-fold decrease in catalytic efficiency for NADH production and a significantly reduced rate of glutaryl-CoA production by dihydrolipoamide succinyl-transferase (E2o). However, the G729R E1a substitution did not affect any of the three side-reactions associated solely with G729R E1a, prompting us to determine the structure-function effects of this mutation. A multipronged systematic analysis of the reaction rates in the OADHc pathway, supplemented with results from chemical cross-linking and hydrogen- deuterium exchange MS, revealed that the c.2185G3A DHTKD1 mutation affects E1a-E2o assembly, leading to impaired channeling of OADHc intermediates. Cross-linking between the C-terminal region of both E1a and G729R E1a with the E2o lipoyl and core domains suggested that correct positioning of the C-terminal E1a region is essential for the intermediate channeling. These findings may inform the development of interventions to counter the effects of pathogenic DHTKD1 mutations. [ABSTRACT FROM AUTHOR]

Abstract Sleep and wakefulness are controlled by a wide range of neuronal populations in the mammalian brain. Activation of adenosine A 2A receptor (A 2A R)-expressing neurons in the nucleus accumbens (NAc) core promotes slow-wave sleep (SWS). The neuronal mechanism by which activation of NAc A 2A R neurons induces SWS, however, is unknown. We hypothesized that the ability of NAc activation to induce sleep is mediated by the classic somnogen adenosine, which can be formed by various processes in all types of cells. Here, to investigate whether astrocytes are involved in the ability of the NAc to regulate SWS, we ablated glial fibrillary acidic protein (GFAP)-positive cells in the NAc core of mice by virus-mediated expression of diphtheria toxin (DT) receptors and intraperitoneal administration of DT. Analysis of electroencephalogram and electromyogram recordings of DT-treated wild-type mice revealed that SWS was remarkably increased at 1 week after DT treatment, whereas sleep-wake behavior was unchanged in DT-treated A 2A R knockout mice. Cell ablation was associated with an increased number of GFAP-positive cells and activation of microglia in the NAc. In-vivo microdialysis revealed significantly increased levels of extracellular adenosine in the NAc at 1 week after DT treatment. Our findings suggest that elevated adenosine levels in the NAc core promote SWS by acting on A 2A Rs and provide the first evidence that adenosine is an endogenous candidate for activating NAc A 2A R neurons that have the ability to induce SWS. Highlights • Ablation of astrocytes and neurons in the nucleus accumbens core increases slow-wave sleep. • Slow-wave sleep is mediated by elevated adenosine levels acting on adenosine A 2A receptors. • Ablation of astrocytes and neurons results in the activation of microglia and astrocytes. [ABSTRACT FROM AUTHOR]

Abstract Insomnia is one of the most common sleep problems with an estimated prevalence of 10%–15% in the general population. Although adenosine A 2A receptor (A 2A R) agonists strongly induce sleep, their cardiovascular effects preclude their use in treating sleep disorders. Enhancing endogenous A 2A R signaling, however, may be an alternative strategy for treating insomnia, because adenosine levels in the brain accumulate during wakefulness. In the present study, we found that 3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid, denoted A 2A R positive allosteric modulator (PAM)-1, enhanced adenosine signaling at the A 2A R and induced slow wave sleep (SWS) without affecting body temperature in wild-type male mice after intraperitoneal administration, whereas the SWS-inducing effect of this benzoic acid derivative was abolished in A 2A R KO mice. In contrast to the A 2A R agonist CGS 21680, the A 2A R PAM-1 did not affect blood pressure or heart rate. These findings indicate that enhancing A 2A R signaling promotes SWS without cardiovascular effects. Therefore, small molecules that allosterically modulate A 2A Rs could help people with insomnia to fall asleep. Graphical abstract Image 1 Highlights • First positive allosteric modulator for adenosine A 2A receptors, denoted A 2A R PAM-1. • A 2A R PAM-1 promotes slow-wave sleep (SWS) in a dose-dependent manner in mice. • Adenosine A 2A receptors are necessary for A 2A R PAM-1 to induce SWS. • Enhancing adenosine A 2A receptor signaling does not induce hypothermia. • Systemic administration of A 2A R PAM-1 does not affect blood pressure or heart rate. [ABSTRACT FROM AUTHOR]

The calcium-binding protein, parvalbumin (PV), is highly expressed in thalamic reticular nucleus (TRN) GABAergic neurons, which receive input from the cerebral cortex and thalamus and send inhibitory output to the thalamic relay nucleus. Previous studies suggest that the TRN is involved in pain regulation as an important relay nucleus of the ascending pain pathway. However, little is known about its functional role in pain regulation and interconnectivity. In our study, the role of rostro-dorsal sector of TRN (TRNrd) PV-positive neurons in pain regulation was studied using chemogenetics based on designer receptors exclusively activated by designer drugs (DREADD). Then, projections from the TRNrd PV-positive neurons were explored using PV-Cre transgenic mice, conditional anterograde axonal tract tracing, and optogenetics, combined with immunohistochemistry and electrophysiology. The results showed that activation of PV-positive neurons in the TRNrd decreased the mechanical threshold and thermal latency of behaving mice during the light period when neuronal activity was low. Furthermore, the anterodorsal and paratenial thalamic nucleus received innervation from PV-positive neurons in the TRNrd. They were specifically inhibited by GABA, which is released from local axonal endings of PV neurons. These findings indicate that activation of PV neurons in the TRNrd increases pain sensitivity in PV-Cre transgenic mice. [ABSTRACT FROM AUTHOR]

Interleukin-1β (IL-1β) and prostaglandin (PG) D 2 are endogenous sleep-promoting substances. Since it was reported that a highly selective cyclooxygenase-2 (COX-2) inhibitor, NS398, completely inhibited IL-1β-induced sleep in rats, IL-1β-induced sleep had been believed to be mediated by prostanoids, most probably PGD 2 . However, in the present study, pretreatment of rats with NS398 (3 mg/kg) did not suppress the 64.2% increased non-rapid eye movement (non-REM, NREM) sleep during infusion of IL-1β (10 ng) for 6 h in the nocturnal (active) period between 23:00 and 5:00 into the subarachnoid space of the PGD 2 -sensitive sleep-promoting zone of the basal forebrain. Meanwhile, IL-1β at doses of 1.7 and 5 μg/kg also significantly increased NREM sleep for 6 h after intraperitoneal injection at 20:00 (light-off time) by 76.8% and 121.1%, respectively, in wild-type (WT) mice, by 67.7% and 147.3%, respectively, in WT mice pretreated with NS398 (5 mg/kg) and by 108.9% and 121.6%, respectively, in PGD 2 receptor (DP 1 R) knockout mice. These results indicate that IL-1β-induced NREM sleep is independent of the PGD 2 /DP 1 R system and other COX-2-derived prostaglandins in rats and mice. [ABSTRACT FROM AUTHOR]

Orexin neurons in the hypothalamus regulate energy homeostasis by coordinating various physiological responses. Past studies have shown the role of the orexin peptide itself; however, orexin neurons contain not only orexin but also other neurotransmitters such as glutamate and dynorphin. In this study, we examined the physiological role of orexin neurons in feeding behavior and metabolism by pharmacogenetic activation and chronic ablation. We generated novel orexin-Cre mice and utilized Cre-dependent adeno-associated virus vectors to express Gq-coupled modified GPCR, hM3Dq or diphtheria toxin fragment A in orexin neurons. By intraperitoneal injection of clozapine-N oxide in orexin-Cre mice expressing hM3Dq in orexin neurons, we could selectively manipulate the activity of orexin neurons. Pharmacogenetic stimulation of orexin neurons simultaneously increased locomotive activity, food intake, water intake and the respiratory exchange ratio (RER). Elevation of blood glucose levels and RER persisted even after locomotion and feeding behaviors returned to basal levels. Accordantly, 83% ablation of orexin neurons resulted in decreased food and water intake, while 70% ablation had almost no effect on these parameters. Our results indicate that orexin neurons play an integral role in regulation of both feeding behavior and metabolism. This regulation is so robust that greater than 80% of orexin neurons were ablated before significant changes in feeding behavior emerged. [ABSTRACT FROM AUTHOR]

Abstract: In humans, a first night effect (FNE) is characterized by increased sleep latency and decreased total sleep time in an unfamiliar environment, but the mechanism and treatment options for this universally experienced acute insomnia are unclear. We continuously recorded electroencephalography (EEG) and electromyogram (EMG) and measured plasma corticosterone levels to develop a mouse FNE model by inducing acute insomnia in mice that have been placed in unfamiliar cage environments. The sleep latency of mice ‘moved to clean cages’ (MCC) was longer than that for mice ‘moved to dirty ones’ (MDC). As compared to MDC mice, MCC mice showed stronger decreases in the amount of non-rapid eye movement (non-REM, NREM) and REM sleep, with a lower power density of NREM sleep, increased fragmentation and decreased stage transitions from NREM sleep to wake, and higher variation in plasma corticosterone levels. Treatment of MCC mice with zolpidem, diazepam, raclopride, pyrilamine, except SCH23390 shortened NREM sleep latency. In addition, zolpidem significantly increased NREM and REM sleep with the increase in slow wave activity (1.00–2.75Hz), while raclopride significantly increased NREM and REM sleep without changing the EEG power density in MCC mice, whereas diazepam increased sleep with a drastic decrease in power density of the frequency band between 1.00 and 4.00Hz, diazepam also increased the frequency band between 9.75 and 24.75Hz during NREM sleep. These results indicate that a MCC mouse can mimic a FNE phenotype of humans and that zolpidem and raclopride may be useful drugs to prevent acute insomnia, including FNE. [Copyright &y& Elsevier]

Trypanosoma brucei prostaglandin F2α synthase is an aldo-ketoreductase that catalyzes the reduction of prostaglandin H2 to PGF2α in addition to that of 9,10-phenanthrenequinone. We report the crystal structure of TbPGFS·NADP+·citrate at 2.1 Å resolution. TbPGFS adopts a parallel (α/β)8-barrel fold lacking the protrudent loops and possesses a hydrophobic core active site that contains a catalytic tetrad of tyrosine, lysine, histidine, and aspartate, which is highly conserved among AKRs. Site-directed mutagenesis of the catalytic tetrad residues revealed that a dyad of Lys77 and His110, and a triad of Tyr52, Lys77, and His110 are essential for the reduction of PGH2 and 9,10-PQ, respectively. Structural and kinetic analysis revealed that His110, acts as the general acid catalyst for PGH2 reduction and that Lys77 facilitates His110 protonation through a water molecule, while exerting an electrostatic repulsion against His110 that maintains the spatial arrangement which allows the formation of a hydrogen bond between His110 and C11 carbonyl of PGH2. We also show that Tyr52 acts as the general acid catalyst for 9,10-PQ reduction, and thus we not only elucidate the catalytic mechanism of a PGH2 reductase but also provide an insight into the catalytic specificity of AKRs. [ABSTRACT FROM AUTHOR]

Vector length as a proxy for the adequacy of ultrafiltration in hemodialysis. Background. Evaluation of dialysis adequacy has focused on parameters of solute (principally urea) clearance. Relatively little attention has been paid to the adequacy of ultrafiltration. At a given phase angle, the bioimpedance vector length reflects the degree of tissue hydration, as the vector lengthens with ultrafiltration. Methods. We determined the relative risk of death associated with different bioimpedance vector lengths in a 3009 patient hemodialysis cohort using proportional hazards regression. Results. The mean phase angle was 4.8°, and the mean vector length 300 ± 70 ohm/m (range 140 to 630 ohm/m). Vector length was much longer in women than men (mean 340 vs. 270 ohm/m) and significantly longer in African Americans and patients without diabetes. Adjusted for the effects of age, gender, race, diabetes, vintage, weight, albumin, prealbumin, creatinine, hemoglobin, ferritin, and dialysis dose, the relative risk (RR) of death was 0.75 (95% CI 0.57 to 0.88) per 100 ohm/m decrease in vector length. The effect of vector length on RR was somewhat more pronounced among men (vector length × gender interaction, P= 0.07). Considering vector length of 300 to 350 ohm/m as the referent category, the RRs of death were 1.54 (95% CI 1.08 to 2.21) and 2.83 (95% CI 1.55 to 5.14) for patients with vector length 200 to 250 and <200 ohm/m, respectively. Conclusion. Shorter predialysis bioimpedance vectors, indicating greater soft tissue hydration, were associated with diminished survival in hemodialysis patients. These findings validate clinical observations linking longevity to maintenance of dry body weight. [ABSTRACT FROM AUTHOR]

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F2α synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH2 to PGF2α. Database search and sequence alignment showed that L. major prostaglandin F2α synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F2α synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F2α synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol. [Copyright &y& Elsevier]

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F2α synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH2 to PGF2α. Database search and sequence alignment alignment showed that L. major prostaglandin F2α synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F2α synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F2α synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol. [Copyright &y& Elsevier]

The olfactory tubercle (OT), an important nucleus in processing sensory information, has been reported to change cortical activity under odor. However, little is known about the physiological role and mechanism of the OT in sleep-wake regulation. The OT expresses abundant adenosine A 2A receptors (A 2A Rs), which are important in sleep regulation. Therefore, we hypothesized that the OT regulates sleep via A 2A Rs. This study examined sleep-wake profiles through electroencephalography and electromyography recordings with pharmacological and chemogenetic manipulations in freely moving rodents. Compared with their controls, activation of OT A 2A Rs pharmacologically and OT A 2A R neurons via chemogenetics increased non-rapid eye movement sleep for 5 and 3 h, respectively, while blockade of A 2A Rs decreased non-rapid eye movement sleep. Tracing and electrophysiological studies showed OT A 2A R neurons projected to the ventral pallidum and lateral hypothalamus, forming inhibitory innervations. Together, these findings indicate that A 2A Rs in the OT play an important role in sleep regulation. • Activation of OT A 2A Rs increased NREM sleep. • Blockade of OT A 2A Rs increased sleep latency. • Chemogenetic activation of OT A 2A R neurons increased NREM sleep. • Connections between OT A 2A R neurons and neurons in the VP and LH were inhibitory. • The study concluded that the OT promotes sleep through A 2A Rs and A2AR neurons. [ABSTRACT FROM AUTHOR]