Brain Injury-Induced Synaptic Reorganization in Hilar Inhibitory Neurons Is Differentially Suppressed by Rapamycin

Following traumatic brain injury (TBI), treatment with rapamycin suppresses mammalian (mechanistic) target of rapamycin (mTOR) activity and specific components of hippocampal synaptic reorganization associated with altered cortical excitability and seizure susceptibility. Reemergence of seizures after cessation of rapamycin treatment suggests, however, an incomplete suppression of epileptogenesis. Hilar inhibitory interneurons regulate dentate granule cell (DGC) activity, and de novo synaptic input from both DGCs and CA3 pyramidal cells after TBI increases their excitability but effects of rapamycin treatment on the injury-induced plasticity of interneurons is only partially described. Using transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed in the somatostatinergic subset of hilar inhibitory interneurons, we tested the effect of daily systemic rapamycin treatment (3 mg/kg) on the excitability of hilar inhibitory interneurons after controlled cortical impact (CCI)-induced focal brain injury. Rapamycin treatment reduced, but did not normalize, the injury-induced increase in excitability of surviving eGFP+ hilar interneurons. The injury-induced increase in response to selective glutamate photostimulation of DGCs was reduced to normal levels after mTOR inhibition, but the postinjury increase in synaptic excitation arising from CA3 pyramidal cell activity was unaffected by rapamycin treatment. The incomplete suppression of synaptic reorganization in inhibitory circuits after brain injury could contribute to hippocampal hyperexcitability and the eventual reemergence of the epileptogenic process upon cessation of mTOR inhibition. Further, the cell-selective effect of mTOR inhibition on synaptic reorganization after CCI suggests possible mechanisms by which rapamycin treatment modifies epileptogenesis in some models but not others

Effects of Rapamycin Treatment on Neurogenesis and Synaptic Reorganization in the Dentate Gyrus after Controlled Cortical Impact Injury in Mice

Post-traumatic epilepsy (PTE) is one consequence of traumatic brain injury (TBI). A prominent cell signaling pathway activated in animal models of both TBI and epilepsy is the mammalian target of rapamycin (mTOR). Inhibition of mTOR with rapamycin has shown promise as a potential modulator of epileptogenesis in several animal models of epilepsy, but cellular mechanisms linking mTOR expression and epileptogenesis are unclear. In this study, the role of mTOR in modifying functional hippocampal circuit reorganization after focal TBI induced by controlled cortical impact (CCI) was investigated. Rapamycin (3 or 10 mg/kg), an inhibitor of mTOR signaling, was administered by intraperitoneal injection beginning on the day of injury and continued daily until tissue collection. Relative to controls, rapamycin treatment reduced dentate granule cell area in the hemisphere ipsilateral to the injury two weeks post-injury. Brain injury resulted in a significant increase in doublecortin immunolabeling in the dentate gyrus ipsilateral to the injury, indicating increased neurogenesis shortly after TBI. Rapamycin treatment prevented the increase in doublecortin labeling, with no overall effect on Fluoro-Jade B staining in the ipsilateral hemisphere, suggesting that rapamycin treatment reduced posttraumatic neurogenesis but did not prevent cell loss after injury. At later times post-injury (8-13 weeks), evidence of mossy fiber sprouting and increased recurrent excitation of dentate granule cells was detected, which were attenuated by rapamycin treatment. Rapamycin treatment also diminished seizure prevalence relative to vehicle-treated controls after TBI. Collectively, these results support a role for adult neurogenesis in PTE development and suggest that suppression of epileptogenesis by mTOR inhibition includes effects on post-injury neurogenesis

Laser Rayleigh Scattering for Flow Density and Condensation Measurements in the Supercooled Mach-14 Free-Stream at AEDC Wind Tunnel No. 9

Laser Rayleigh scattering was used to investigate clusters in the free-stream flow at Arnold Engineering Development Centers Tunnel 9 (T9). The facility was run at Mach-14, with a pure-N2 flow medium, and at several total pressures and temperatures. Using an excimer laser operating at 248 nm, the Rayleigh instrument imaged scattering from the focused laser beam in the free-stream. As a wind-tunnel flow is accelerated, it cools and approaches the condensation boundary. As a precursor to condensation, small clusters of molecules are first formed, but the individual clusters are too small to be spatially resolved in typical images of the beam. Thus clusters effectively add a spatially smooth background signal to the pure diatomic-molecule Rayleigh signal. The main result of the present work is that clustering was not significant. After correcting for interference by small particles imbedded in the T9 flow, cluster scattering was unobservable or smaller than one standard deviation (1-sigma) of the uncertainties for almost all tunnel runs. The total light scattering level was measured to be 1.05 +/- 0.15 (1-sigma) of the expected diatomic scattering, when averaged over the entire usable data set. This result included flow conditions that were supercooled to temperatures of ~ 20 K, about 25 K below the condensation limit of ~ 45 K. Thus the Mach-14 nozzle flow is essentially cluster-free for many supercooled conditions that might be used to extend the facility operating range to larger Reynolds numbers

Challenges of Incorporating Digital Health Technology Outcomes in a Clinical Trial: Experiences from PD STAT.

Digital health technologies (DHTs) have great potential for use as clinical trial outcomes; however, practical issues need to be addressed in order to maximise their benefit. We describe our experience of incorporating two DHTs as secondary/exploratory outcome measures in PD STAT, a randomised clinical trial of simvastatin in people with Parkinson's disease. We found much higher rates of missing data in the DHTs than the traditional outcome measures, in particular due to technical and software difficulties. We discuss methods to address these obstacles in terms of protocol design, workforce training and data management

Challenges of Incorporating Digital Health Technology Outcomes in a Clinical Trial: Experiences from PD STAT

\ua9 2022 - The authors. Published by IOS Press.Digital health technologies (DHTs) have great potential for use as clinical trial outcomes; however, practical issues need to be addressed in order to maximise their benefit. We describe our experience of incorporating two DHTs as secondary/exploratory outcome measures in PD STAT, a randomised clinical trial of simvastatin in people with Parkinson\u27s disease. We found much higher rates of missing data in the DHTs than the traditional outcome measures, in particular due to technical and software difficulties. We discuss methods to address these obstacles in terms of protocol design, workforce training and data management

Mortality of Cranes (Gruidae) Associated with Powerlines over a Major Roost on the Platte River, Nebraska

Two 69-kilovolt powerlines spanning the Platte River in south central Nebraska are suspected to cause substantial mortality to sandhill cranes (Grus canadensis) and pose a threat to endangered whooping cranes (G. americana) that roost overnight on the river during spring and fall migrations. Most studies of crane collisions with powerlines in the region have focused on counts of carcasses away from night roosts on the river and none have accounted for potential biases in detecting carcasses. We found 61 carcasses of sandhill cranes below over-river segments of the two powerlines during 4 March to 7 April 2006 and 90 such carcasses between 5 March and 13 April 2007. In 2007 we estimated the number of carcasses undetected in our surveys due to removal by scavengers, loss to downstream flow, and observer oversight. We estimated between 165 and 219 sandhill cranes were killed by the two powerlines during spring 2007. These cnlculations exclude mortalities from individuals injured by powerline collisions and dying elsewhere, as well as those killed before or after our 5 March to 13 April survey period. We detected no evidence of mortality for whooping cranes during our surveys. Our results corroborate anecdotal evidence of signficant sandhill crane mortality each spring due to collisions with above-ground powerlines at this major night roost. Collisions by sandhill cranes will continue and collisions by Whooping cranes seem likely unless an effective means of averting birds from powerlines is implemented at this site

Quality assurance of 61Cu using ICP mass spectroscopy and metal complexation

Introduction 61Cu (T1/2 = 3.33 hr, Eβ= 1.22 MeV, 61.4 %) is an attractive isotope for positron emission tomography (PET) radiopharmaceutical agents such as ATSM and PTSM. Various separation processes have been reported for the production of 61Cu on a medium cyclotron using 13–22 MeV protons on natural and enriched 64Zn target materials [1,2]. This work, investigates production of 61Cu using both natural and enriched 64Zn targets and its separation. Three types of resins were used to assess for their efficiency and speed to separate the desired 61Cu from the 66,67,68Ga and 64Zn and for the recycling of 64Zn target material. The effective specific activity of purified 61Cu, was determined by ICP-MS and its titration with various polyaza and polycarboxylate complexing ligands. Material and Methods 1. Production and Separation Targets were irradiated by proton beam of IBA cyclotron 18/18MeV via the 64Zn(p,α) 61Cu and natZn(p,x) 61Cu reactions using an enriched 64Zn foil(15×15×0.05mm, ~50 mg) and natural foil (diameter 25 mm, 0.05 mm,~ 60 mg). Thirty minute irradiations were conducted with incident proton energies between 11.7–12.0 MeV and beam currents of 20 and 40 µA. Irradiated Zn targets were dissolved in 8M HCl at 150 oC then evaporated to dryness. Trace water to the resultant residue (twice) and resultant solutions evaporated to dryness. The residue was re-dissolved in 2ml of 0.01M HCl before loading onto a Cu-resin column (FIG. 1) Zn and Ga isotopes were collectively eluted using 30 ml of 0.01M HCl. The Cu was then removed using 1.5 ml of 8M HCl and passed directly onto a cation exchange followed by an anion exchange column. An additional 3 ml of 8M HCl was used to rinse the cation exchange column and ensure quantitatively removal of Cu (II) ions. The Cu was finally eluted from the anion exchange column using 3 ml of 2M HCl. The Cu solution was heated up at 150 oC until evaporated to dryness and 61Cu final product dissolved in 400–800 μL of 0.01M HCl. 2. Specific activity of 61Cu The specific activity (GBq/µmol) of the purified 61Cu was determined by ICP-MS and compared with that determined using dota, nota and di-amsar complexing ligands. For each 61Cu production run aliquot of final solution (100 µL) was left to decay before dilut-ing to 10 mL with 10% HNO3. Decayed samples were sent to ChemCentre (Curtin University) for ICP-MS analysis. Each sample was analysed for Cu, Al, Ca, Co, Fe, Ga, Ni, Si, and Zn, which are known to compete with Cu2+ for ligand complexation. Effective specific activity of the 61Cu was deter-mined by titrating various known concentration of ligands with 61Cu solution. The method is detailed in the literature [3]. Briefly, varying concentrations of each ligand was prepared in 0.1M sodium acetate buffer pH 6.5 to a total volume 20 µL. Fixed concentration of diluted 61Cu (0.01M HCl) in 10 µL was added to each ligand solution. The mixtures were vortexed then left to incubate at the room temperature for 30 mins. Two uL aliquots were withdrawn (in triplicate) from each reaction mixture and spot-ted on ITLC –SA. [Mobile phase: 0.1M NaCl: 0.1M EDTA (9:1) for Cu2+ and diamsar mixtures: Rf 0.8 free Cu2+ and 0.1M sodium acetate pH 4.5: H2O: MeOH: ammonium hydroxide (20:18:2:1 v/v) for Cu2+ dota and nota mixtures: Rf >0.8 Cu-dota and Cu-nota Rf < 0.2 free Cu2+]. Complexation of the 61Cu with each ligand was complete within 30 mins at room temperature. Concentration of Cu2+ was deter-mined from the 50% labelling efficiency. Results and Conclusion 1. Production and Separation The radioisotopes production from natZn target must be minimized by the optimum proton energy to reduce a radiation dose in the final product. The excitation functions of 66,67,68Ga ,65Zn and 61Cu are shown in FIG. 2. Proton beam energy of 11.7 MeV was used for both Zn targets to minimise the production of Ga isotopes and prevent formation of 65Zn. For the enriched 64Zn target (99.30%) higher proton energy could be used for the production of 61Cu allowing for increased yields and reduce radio contaminants. Previously, we used anion and cation exchange resin as described in the literature to separate the 61Cu [1]. Unfortunately the literature method was too long (up to 3 hours) and requiring high concentration of HCl and long evaporation times compromising achievable yields [4]. Thieme S. et al., 2013 [2] reported the successful use of Cu-resin for the separation of Cu radioisotopes and it was of interest to the current work to test this material for the separation of 61Cu in our hands. A cation, anion exchange and Cu-resin were combined into closed system to separate the 61Cu within 30 mins (FIG. 1). The system is designed to contain the transfer of solutions be-tween each column using simple plunger to force solution through and between each column. This system afforded an easy, reliable and fast separation of 61Cu that could be completed within 30 min. 2. Specific activity The specific activity of 61Cu was determined using ICP-MS and by titration with three ligands is summarized in TABLE 1. The ICP-MS data show values ranging from 9.2 to 32.4 GBq/μmol for 8 production runs. Specific activity determine using nota and dota were in all cases lower than the ICP MS data indicating some interference from the other metal ion contaminates such as Fe(ii/Iii), Ni (II), Ca (II), Zn (II), Ga (III). The specific activity determine using diamsar, which is known to be highly selective for Cu(II) (and Zn(II) and Fe(III)) in the presence of alkali and alkaline earth ions gave values significantly higher effective specific activity than that obtained using ICP MS. Variations in values can be explained by presence of contaminating metal ions

A Hindbrain Inhibitory Microcircuit Mediates Vagally-Coordinated Glucose Regulation

Neurons in the brainstem dorsal vagal complex integrate neural and humoral signals to coordinate autonomic output to viscera that regulate a variety of physiological functions, but how this circuitry regulates metabolism is murky. We tested the hypothesis that premotor, GABAergic neurons in the nucleus tractus solitarius (NTS) form a hindbrain micro-circuit with preganglionic parasympathetic motorneurons of the dorsal motor nucleus of the vagus (DMV) that is capable of modulating systemic blood glucose concentration. In vitro, neuronal activation or inhibition using either excitatory or inhibitory designer receptor exclusively activated by designer drugs (DREADDs) constructs expressed in GABAergic NTS neurons increased or decreased, respectively, action potential firing of GABAergic NTS neurons and downstream synaptic inhibition of the DMV. In vivo, DREADD-mediated activation of GABAergic NTS neurons increased systemic blood glucose concentration, whereas DREADD-mediated silencing of these neurons was without effect. The DREADD-induced hyperglycemia was abolished by blocking peripheral muscarinic receptors, consistent with the hypothesis that altered parasympathetic drive mediated the response. This effect was paralleled by elevated serum glucagon and hepatic phosphoenolpyruvate carboxykinase 1 (PEPCK1) expression, without affecting insulin levels or muscle metabolism. Activity in a hindbrain inhibitory microcircuit is sufficient to modulate systemic glucose concentration, independent of insulin secretion or utilization

NCS-1 Inhibits insulin stimulated GLUT4 translocation in 3T3L1 adipocytes through a phosphatidylinositol 4-kinase dependent pathway

Expression of NCS-1 (neuronal calcium sensor-1, also termed frequenin) in 3T3L1 adipocytes strongly inhibited insulin-stimulated translocation of GLUT4 and insulin-responsive aminopeptidase. The effect of NCS-1 was specific for GLUT4 and the insulin-responsive aminopeptidase translocation as there was no effect on the trafficking of the cation-independent mannose 6-phosphate receptor or the GLUT1 glucose transporter isoform. Moreover, NCS-1 showed partial colocalization with GLUT4-EGFP in the perinuclear region. The inhibitory action of NCS-1 was independent of calcium sequestration since neither treatment with ionomycin nor endothelin-1, both of which elevated the intracellular calcium concentration, restored insulin-stimulated GLUT4 translocation. Furthermore, NCS-1 did not alter the insulin-stimulated protein kinase B (PKB/Akt) phosphorylation or the recruitment of Cbl to the plasma membrane. In contrast, expression of the NCS-1 effector phosphatidylinositol 4-kinase (PI 4-kinase) inhibited insulin-stimulated GLUT4 translocation, whereas co-transfection with an inactive PI 4-kinase mutant prevented the NCS-1-induced inhibition. These data demonstrate that PI 4-kinase functions to negatively regulate GLUT4 translocation through its interaction with NCS-1