Pedunculopontine arousal system physiology – Implications for insomnia

AbstractWe consider insomnia a disorder of waking rather than a disorder of sleep. This review examines the role of the reticular activating system, especially the pedunculopontine nucleus, in the symptoms of insomnia, mainly representing an overactive waking drive. We determined that high frequency activity during waking and REM sleep is controlled by two different intracellular pathways and channel types in PPN cells. We found three different PPN cell types that have one or both channels and may be active during waking only, REM sleep only, or both. These discoveries point to a specific mechanism and novel therapeutic avenues for insomnia

hiPSC-Based Tissue Organoid Regeneration

Induced pluripotent stem cells (iPSCs) are generated from terminally differentiated cells and have the potential to differentiate to any organs originated from the embryonic germ layers. Extensive effort has been made to establish protocols for direct in vitro conversion of human iPSCs (hiPSCs) to different cell types/organs. Importantly, hiPSCs can be generated from patients with known genetic mutations that predispose to high-risks of specific disease development. Thus, the hiPSCs technology provides unlimited resources for creating patient-specific disease models. hiPSC-derived three-dimensional “organoid” models have recently emerged as a powerful tool to recapitulate the physiologically-relevant process of disease progression in vitro. In this chapter, we will discuss the current advancement of organoid regeneration from hiPSCs and the applications of hiPSCs-derived organoids. The limitations and challenges of this approach will also be discussed here

Muscarinic Modulation of High Frequency Oscillations in Pedunculopontine Neurons

We previously reported that persistent application of the non-specific cholinergic agonist carbachol (CAR) increased the frequency of calcium channel-mediated oscillatory activity in pedunculopontine nucleus (PPN) neurons, which we identified as dependent on voltage-gated, high-threshold P/Q-type channels. Here, we tested the hypothesis that M2 muscarinic receptors and G-proteins associated with M2 receptors mediate the increase in oscillatory frequency in PPN neurons. We found, using depolarizing ramps, that patch clamped 9–12 day old rat PPN neurons (n = 189) reached their peak oscillatory activity around −20 mV membrane potential. Acute (short duration) application of CAR blocked the oscillatory activity through M2 muscarinic receptors, an effect blocked by atropine. However, persistent (long duration) application of CAR significantly increased the frequency of oscillatory activity in PPN neurons through M2 receptors [40 ± 1 Hz (with CAR) vs. 23 ± 1 Hz (without CAR); p < 0.001]. We then tested the effects of the G-protein antagonist guanosine 5′-[β-thio] diphosphate trilithium salt (GDP-β-S), and the G-protein agonist 5′-[γ-thio] triphosphate trilithium salt (GTP-γ-S). We found, using a three-step protocol in voltage-clamp mode, that the increase in the frequency of oscillations induced by M2 cholinergic receptors was linked to a voltage-dependent G-protein mechanism. In summary, these results suggest that persistent cholinergic input creates a permissive activation state in the PPN that allows high frequency P/Q-type calcium channel-mediated gamma oscillations to occur

The American Dream eludes many urban youth of color. And they think it's their own fault.

Segregation is not a thing of the past for young people of color in the US; many attend under-resourced and segregated school districts. Despite these disadvantages, America's leaders tell young people of color that they can achieve the "American Dream" via hard work and determination. In new research, David T. Lardier Jr., Kathryn G. Herr, Veronica R. Barrios, Pauline Garcia-Reid ..

Activated Bone Marrow-Derived Macrophages Eradicate Alzheimer's-Related Aβ42 Oligomers and Protect Synapses.

Impaired synaptic integrity and function due to accumulation of amyloid β-protein (Aβ42) oligomers is thought to be a major contributor to cognitive decline in Alzheimer's disease (AD). However, the exact role of Aβ42 oligomers in synaptotoxicity and the ability of peripheral innate immune cells to rescue synapses remain poorly understood due to the metastable nature of oligomers. Here, we utilized photo-induced cross-linking to stabilize pure oligomers and study their effects vs. fibrils on synapses and protection by Aβ-phagocytic macrophages. We found that cortical neurons were more susceptible to Aβ42 oligomers than fibrils, triggering additional neuritic arborization retraction, functional alterations (hyperactivity and spike waveform), and loss of VGluT1- and PSD95-excitatory synapses. Co-culturing neurons with bone marrow-derived macrophages protected synapses against Aβ42 fibrils; moreover, immune activation with glatiramer acetate (GA) conferred further protection against oligomers. Mechanisms involved increased Aβ42 removal by macrophages, amplified by GA stimulation: fibrils were largely cleared through intracellular CD36/EEA1+-early endosomal proteolysis, while oligomers were primarily removed via extracellular/MMP-9 enzymatic degradation. In vivo studies in GA-immunized or CD115+-monocyte-grafted APPSWE/PS1ΔE9-transgenic mice followed by pre- and postsynaptic analyses of entorhinal cortex and hippocampal substructures corroborated our in vitro findings of macrophage-mediated synaptic preservation. Together, our data demonstrate that activated macrophages effectively clear Aβ42 oligomers and rescue VGluT1/PSD95 synapses, providing rationale for harnessing macrophages to treat AD

Proteomic measures of gamma oscillations

Background: Gamma oscillations serve complex processes, and the first stage of their generation is the reticular activating system (RAS), which mediates the gamma-activity states of waking and paradoxical sleep. We studied whether the pedunculopontine nucleus (PPN), part of the RAS in which every cell manifests intrinsic gamma oscillations, undergoes changes resulting in distinctive protein expression. New method: We previously found that a histone deacetylation inhibitor, trichostatin A (TSA), acutely (30 min) blocked these oscillations. We developed a proteomic method for sampling stimulated and unstimulated PPN and determining protein expression in 1 mm punches of tissue from brain slices subjected to various treatments. Results: We compared brain slices exposed for 30 min to TSA (unstimulated), to the cholinergic agonist carbachol (CAR), known to induce PPN gamma oscillations, or exposed to both TSA + CAR. Comparison with existing methods: Label-free proteomics provides an unbiased and sensitive method to detect protein changes in the PPN. Our approach is superior to antibody-based methods that can lack specificity and can only be done for known targets. Proteomics methods like these have been leveraged to study molecular pathways in numerous systems and disease states. Conclusions: Significant protein changes were seen in two functions essential to the physiology of the PPN: cytoskeletal and intracellular [Ca2+] regulation proteins. TSA decreased, while CAR increased, and TSA + CAR had intermediate effects, on expression of these proteins. These results support the feasibility of the methods developed for determining proteomic changes in small samples of tissue participating in the most complex of brain processes.Fil: Byrum, Stephanie D.. Arkansas Children's Research Institute; Estados UnidosFil: Washam, Charity L.. Arkansas Children's Research Institute; Estados UnidosFil: Tackett, Alan J.. Arkansas Children's Research Institute; Estados UnidosFil: Garcia Rill, Edgar. University of Arkansas for Medical Sciences; Estados UnidosFil: Bisagno, Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Urbano Suarez, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Engineered LINE-1 retrotransposition in nondividing human neurons

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80-100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought.We thank current members of the J.L.G.-P. laboratory for helpful discussions. We also thank Drs. Geoffrey Faulkner (Mater Research, Australia) and John V. Moran (University of Michigan) for sharing unpublished data and for critical input during the project; Ms. Raquel Marrero (Microscopy Unit, Genyo) for technical support; Simon Mendez-Ferrer (CNIC, Spain) for providing total RNA isolated from human mesenspheres; Dr. Oliver Weichenrieder (Max-Planck, Tubingen, Germany) for providing a polyclonal L1-ORF1p antibody; and Dr. Aurelien Doucet (IRCAN, Nice, France) for providing a plasmid containing an UBC-driven EGFP retrotransposition indicator cassette. J.L.G. was funded by the US Department of Defense, Breast Cancer Research Program (award #BC051386), the National Institutes of Health (NIH) National Institute of Neurological Disorders and Stroke (1R03NS087290-01), and the ALS Therapy Alliance (2013-F-067). A.M. has been partially funded by a Marie Curie IRG project (FP7-PEOPLE-2007-4-3-IRG: SOMATIC LINE-1). J.L.G.-P's laboratory is supported by CICE-FEDER-P09-CTS-4980, CICE-FEDER-P12-CTS-2256, Plan Nacional de I+D+I 2008–2011 and 2013–2016 (FIS-FEDER-PI11/01489 and FIS-FEDER-PI14/02152), PCIN-2014-115-ERA-NET NEURON II, the European Research Council (ERC-Consolidator ERC-STG-2012-233764), by an International Early Career Scientist grant from the Howard Hughes Medical Institute (IECS-55007420), and by The Wellcome Trust–University of Edinburgh Institutional Strategic Support Fund (ISFF2).S

Symbolic dynamics to enhance diagnostic ability of portable oximetry from the Phone Oximeter in the detection of paediatric sleep apnoea

Objective: This study is aimed at assessing symbolic dynamics as a reliable technique to characterize complex fluctuations of portable oximetry in the context of automated detection of childhood obstructive sleep apnoea-hypopnoea syndrome (OSAHS). Approach: Nocturnal oximetry signals from 142 children with suspected OSAHS were acquired using the Phone Oximeter: a portable device that integrates a pulse oximeter with a smartphone. An apnoea-hypopnoea index (AHI) ⩾ 5 events h−1 from simultaneous in-lab polysomnography was used to confirm moderate-to-severe childhood OSAHS. Symbolic dynamics was used to parameterise non-linear changes in the overnight oximetry profile. Conventional indices, anthropometric measures, and time-domain linear statistics were also considered. Forward stepwise logistic regression was used to obtain an optimum feature subset. Logistic regression (LR) was used to identify children with moderate-to-severe OSAHS. Main results: The histogram of 3-symbol words from symbolic dynamics showed significant differences (p &lt; 0.01) between children with AHI &lt; 5 events h−1 and moderate-to-severe patients (AHI ⩾ 5 events h−1). Words representing increasing oximetry values after apnoeic events (re-saturations) showed relevant diagnostic information. Regarding the performance of individual characterization approaches, the LR model composed of features from symbolic dynamics alone reached a maximum performance of 78.4% accuracy (65.2% sensitivity; 86.8% specificity) and 0.83 area under the ROC curve (AUC). The classification performance improved combining all features. The optimum model from feature selection achieved 83.3% accuracy (73.5% sensitivity; 89.5% specificity) and 0.89 AUC, significantly (p &lt;0.01) outperforming the other models. Significance: Symbolic dynamics provides complementary information to conventional oximetry analysis enabling reliable detection of moderate-to-severe paediatric OSAHS from portable oximetry