Modulation of nicotinic receptors by an allosteric ligand assessed using fast jumps in acetylcholine

Thesis (M.S.) University of Alaska Fairbanks, 2016Properties of ligand-gated ion channels such as α4β2 nicotinic acetylcholine receptors (nAChRs) and their interactions with various pharmacologic compounds have been studied using voltage clamping techniques for decades. The peak current amplitude, measured in whole-cell experiments, gives us an idea of how receptors will respond to a ligand in situ. Some ligands have the potential to potentiate the peak amplitude, by various mechanisms such as destabilization of receptor desensitized states. The ability of a ligand to increase the peak amplitude of α4β2 nAChRs has the potential to treat a variety of neuronal disorders; however unique properties of these receptors such as fast activation and long-lived desensitized states create significant challenges in determining the extent of modulation of the peak current using these techniques. To correctly assess the peak amplitude, the experiment must achieve synchronous activation of all surface receptors by optimizing solution exchange rates. Failure to do so leads to blunted peak-amplitude measurements in acetylcholine (ACh). This study found that previous reports of the modulating effects of desformylflustrabromine (dFBr), a positive allosteric modulator (PAM) of α4β2 nAChRs, neglected to account for the large surface area of Xenopus oocytes and slow solution exchange rates, leading to an artificially large potentiation of the peak current in dFBr. This study utilized cell lines with a relatively small surface area and a high-speed perfusion system to achieve fast solution exchange rates, and found the potentiation of the peak current by dFBr to be ~1.5-fold. Further studies involving PAMs of α4β2 nAChRs should take necessary steps to optimize solution exchange rates to improve accuracy and reproducibility of their results. In addition, analysis of the whole-cell responses of α4β2 nAChRs to dFBr and Ach have lead to new insights on their effect on not only the peak amplitude, but also on the time-to peak, and the steady-state current. On average, we found that dFBr decreased the time-to peak by 38% and increased the steady-state current ~1.5-fold. Further studies should also consider modulation of the steady-state current to be just as, if not more important than the peak amplitude, as this feature may be a better predictor of the therapeutic benefit of PAMs of α4β2 nAChRs

An episode mimicking a versive seizure in acute bilateral pontine stroke.

Pontine ischemia usually results in focal deficits such as hemiparesis, facial palsy, dysarthria, disorders of eye movements or vertigo. Although rarely described, involuntary abnormal movements and "convulsions" due to pontine lesions can also occur. Here we describe a 67-year-old woman with hypertension who presented with a tonic movement mimicking a versive seizure in the acute phase of bilateral pontine ischemia. Post-stroke movement disorders are well known. They are usually associated with supratentorial lesions and rarely occur in the acute phase, but "seizure-like" episodes can be seen in pontine ischemia. Awareness of this rare phenomenon is useful for the management of acute stroke patients

Cell-specific modulation of monocarboxylate transporter expression contributes to the metabolic reprograming taking place following cerebral ischemia.

Monocarboxylate transporters (MCTs) are involved in lactate trafficking and utilization by brain cells. As lactate is not only overproduced during ischemia but its utilization was shown to be essential upon recovery, we analyzed the expression of the main cerebral MCTs at 1 and 24h after an ischemic insult induced by a transient occlusion of the left middle cerebral artery (MCAO) in CD1 mice (n=5, 7 and 10 for control, 1 and 24h groups, respectively). After 1h of reperfusion, an upregulation of the three MCTs was observed in the striatum (MCT1 ipsilateral 2.73 ± 0.2 and contralateral 2.01 ± 0.4; MCT2 ipsilateral 2.1 ± 0.1; MCT4 ipsilateral 1.65 ± 0.1) and in the surrounding cortex of both the ipsilateral (MCT1 2.4 ± 0.4; MCT2 1.62 ± 0.2; MCT4 1.31 ± 0.1) and contralateral (MCT1 2.78 ± 0.4; MCT2 1.76 ± 0.2) hemispheres, compared to the corresponding sham hemispheres. An increase of MCT1 (ipsilateral 2.1 ± 0.2) and MCT2 (contralateral 1.9 ± 0.1) expression was also observed in the hippocampus, while no effect was observed for MCT4. At 24h of reperfusion, total MCT2 and MCT4 expressions were decreased in the striatum (MCT2 ipsilateral 0.32 ± 0.1 and contralateral 0.63 ± 0.1; MCT4 ipsilateral 0.59 ± 0.1) and the surrounding cortex (MCT4 ipsilateral 0.67 ± 0.1), compared to the sham. At the cellular level, neurons which usually express only MCT2 strongly expressed MCT1 at both time points. Surprisingly, staining for MCT4 appeared on neurons and was strong at 24h post-insult, in the striatum and the cortex of both hemispheres. A similar expression pattern was observed also in the ipsilateral hemisphere of the sham operated animals at 24h. Overall, our study indicates that cell-specific changes in MCT expression induced by an ischemic insult may participate to the metabolic adaptations taking place in the brain after a transient ischemic episode

Spatio-temporal overview of neuroinflammation in an experimental mouse stroke model.

After ischemic stroke, in the lesion core as well as in the ischemic penumbra, evolution of tissue damage and repair is strongly affected by neuroinflammatory events that involve activation of local specialized glial cells, release of inflammatory mediators, recruiting of systemic cells and vascular remodelling. To take advantage of this intricate response in the quest to devise new protective therapeutic strategies we need a better understanding of the territorial and temporal interplay between stroke-triggered inflammatory and cell death-inducing processes in both parenchymal and vascular brain cells. Our goal is to describe structural rearrangements and functional modifications occurring in glial and vascular cells early after an acute ischemic stroke. Low and high scale mapping of the glial activation on brain sections of mice subjected to 30 minutes middle cerebral artery occlusion (MCAO) was correlated with that of the neuronal cell death, with markers for microvascular changes and with markers for pro-inflammatory (IL-1β) and reparative (TGFβ1) cytokines. Our results illustrate a time-course of the neuroinflammatory response starting at early time-points (1 h) and up to one week after MCAO injury in mice, with an accurate spatial distribution of the observed phenomena

Hydroxycarboxylic Acid Receptor 1 and Neuroprotection in a Mouse Model of Cerebral Ischemia-Reperfusion.

Lactate is an intriguing molecule with emerging physiological roles in the brain. It has beneficial effects in animal models of acute brain injuries and traumatic brain injury or subarachnoid hemorrhage patients. However, the mechanism by which lactate provides protection is unclear. While there is evidence of a metabolic effect of lactate providing energy to deprived neurons, it can also activate the hydroxycarboxylic acid receptor 1 (HCAR1), a Gi-coupled protein receptor that modulates neuronal firing rates. After cerebral hypoxia-ischemia, endogenously produced brain lactate is largely increased, and the exogenous administration of more lactate can decrease lesion size and ameliorate the neurological outcome. To test whether HCAR1 plays a role in lactate-induced neuroprotection, we injected the agonists 3-chloro-5-hydroxybenzoic acid and 3,5-dihydroxybenzoic acid into mice subjected to 30-min middle cerebral artery occlusion. The in vivo administration of HCAR1 agonists at reperfusion did not appear to exert any relevant protective effect as seen with lactate administration. Our results suggest that the protective effects of lactate after hypoxia-ischemia come rather from the metabolic effects of lactate than its signaling through HCAR1

Teacher Use of Diagnostic Score Reports for Instructional Decision-Making in the Subsequent Academic Year

Large-scale summative assessment results are typically used for program-evaluation and resource-allocation purposes; however, stakeholders increasingly desire results from large-scale K–12 assessments that inform instruction. Because large-scale summative results are usually delivered after the end of the school year, teacher use of results is reserved for the subsequent academic year. To evaluate use of summative score reports to inform instruction, we conducted a series of teacher interviews and focus groups with 17 teachers in three states. Teachers were asked to describe how they used summative results from the previous administration of a large-scale alternate assessment system in the subsequent academic year. Interview and focus-group transcripts were coded and identified themes related to when and how score reports are delivered; how teachers use results to plan instruction, formulate goals for individualized education programs (IEPs), and create instructional groupings; how teachers talk to parents about results; and what resources best support their use of score reports. Findings demonstrate preliminary support for diagnostic score report use

Non-invasive diagnostic biomarkers for estimating the onset time of permanent cerebral ischemia.

The treatments for ischemic stroke can only be administered in a narrow time-window. However, the ischemia onset time is unknown in ~30% of stroke patients (wake-up strokes). The objective of this study was to determine whether MR spectra of ischemic brains might allow the precise estimation of cerebral ischemia onset time. We modeled ischemic stroke in male ICR-CD1 mice using a permanent middle cerebral artery filament occlusion model with laser Doppler control of the regional cerebral blood flow. Mice were then subjected to repeated MRS measurements of ipsilateral striatum at 14.1 T. A striking initial increase in γ-aminobutyric acid (GABA) and no increase in glutamine were observed. A steady decline was observed for taurine (Tau), N-acetyl-aspartate (NAA) and similarly for the sum of NAA+Tau+glutamate that mimicked an exponential function. The estimation of the time of onset of permanent ischemia within 6 hours in a blinded experiment with mice showed an accuracy of 33±10 minutes. A plot of GABA, Tau, and neuronal marker concentrations against the ratio of acetate/NAA allowed precise separation of mice whose ischemia onset lay within arbitrarily chosen time-windows. We conclude that (1)H-MRS has the potential to detect the clinically relevant time of onset of ischemic stroke

Involvement of caveolin-1 in neurovascular unit remodeling after stroke: Effects on neovascularization and astrogliosis.

Complex cellular and molecular events occur in the neurovascular unit after stroke, such as blood-brain barrier (BBB) dysfunction and inflammation that contribute to neuronal death, neurological deterioration and mortality. Caveolin-1 (Cav-1) has distinct physiological functions such as caveolae formation associated with endocytosis and transcytosis as well as in signaling pathways. Cav-1 has been proposed to be involved in BBB dysfunction after brain injury; however, its precise role is poorly understood. The goal of this study was to characterize the expression and effect of Cav-1 deletion on outcome in the first week in a transient Middle Cerebral Artery Occlusion stroke model. We found increased Cav-1 expression in new blood vessels in the lesion and in reactive astrocytes in the peri-lesion areas. In Cav-1 KO mice, the lesion volume was larger and the behavioral outcome worse than in WT mice. Cav-1 KO mice exhibited reduced neovascularization and modified astrogliosis, without formation of a proper glial scar around the lesion at three days post injury, coinciding with aggravated outcomes. Altogether, these results point towards a potential protective role of endogenous Cav-1 in the first days after ischemia by promoting neovascularization, astrogliosis and scar formation

Evaluating the potential of hyperpolarised [1-13C] L-lactate as a neuroprotectant metabolic biosensor for stroke.

Cerebral metabolism, which can be monitored by magnetic resonance spectroscopy (MRS), changes rapidly after brain ischaemic injury. Hyperpolarisation techniques boost <sup>13</sup> C MRS sensitivity by several orders of magnitude, thereby enabling in vivo monitoring of biochemical transformations of hyperpolarised (HP) <sup>13</sup> C-labelled precursors with a time resolution of seconds. The exogenous administration of the metabolite L-lactate was shown to decrease lesion size and ameliorate neurological outcome in preclinical studies in rodent stroke models, as well as influencing brain metabolism in clinical pilot studies of acute brain injury patients. The aim of this study was to demonstrate the feasibility of measuring HP [1- <sup>13</sup> C] L-lactate metabolism in real-time in the mouse brain after ischaemic stroke when administered after reperfusion at a therapeutic dose. We showed a rapid, time-after-reperfusion-dependent conversion of [1- <sup>13</sup> C] L-lactate to [1- <sup>13</sup> C] pyruvate and [ <sup>13</sup> C] bicarbonate that brings new insights into the neuroprotection mechanism of L-lactate. Moreover, this study paves the way for the use of HP [1- <sup>13</sup> C] L-lactate as a sensitive molecular-imaging biosensor in ischaemic stroke patients after endovascular clot removal

Crystallographic—magnetic correlations in single-crystal haemo-ilmenite: new evidence for lamellar magnetism

17 single crystals were identified by electron backscatter diffraction (EBSD) and isolated from coarse massive haemo-ilmenite ore from South Rogaland, Norway. These were studied using the EBSD results, natural remanent magnetization (NRM), and anisotropy of magnetic susceptibility (AMS), to gain a better understanding of angular relationships between crystallographic axes and magnetic properties of haemo-ilmenite in relation to lamellar magnetism. Electron microprobe analyses gave the following average end-member compositions for ilmenite host: 21.1 per cent MgTiO3, 73.7 FeTiO3, 0.5 MnTiO3, 4.3 Fe2O3, 0.2 Cr2O3 and 0.3 V2O3; and for the coarsest (∼3 μm) haematite exsolution lamellae: 3.5 MgTiO3, 22.4 FeTiO3, 71.4 Fe2O3, 1.6 Cr2O3, 1.0 V2O3 and 0.1 Al2O3, making this sample the most Mg- and Cr-rich haemo-ilmenite studied in the province, but with similar element fractionations between the coexisting phases. TEM work on similar material suggests the presence of much thinner exsolution down to 1-2 nm. The EBSD, NRM and AMS results from 12 out of 17 crystals indicate a good agreement between the orientation of crystallographic axes, NRM direction and principal axes of the magnetic susceptibility ellipsoid, with the NRM located in the (0001) basal plane [NRM ∧ (0001) < 6.5°] and the crystallographic c axis quasi-parallel to the minimum axis of the susceptibility ellipsoid [c∧ k3 < 13.5°]. In addition, in 10 of these 12 crystals, the remanent magnetization vector is parallel or nearly parallel to the positive direction of a crystallographic a axis [NRM ∧a < 20°], hence parallel to a principal magnetic moment direction in haematite as determined by Besser, and not parallel to the spin-canted direction of end-member haematite. This is consistent with a basic property of lamellar magnetism, where the magnetic moment is parallel to the principal moments (sublattice magnetization directions) in haematite. Relationships in three additional crystals with NRM ∧a= 22°-33°, only two with good agreement, can be interpreted as consistent with having a magnetic vector quasi-parallel to the spin-canted direction of haematit