Robia G. Pautler

Tracing odor-induced activation in the olfactory bulbs of mice using manganese-enhanced magnetic resonance imaging.

Ithas previously been demonstrated that it is possible to map active regions of the brain using MRI relying on the fact that Mn(2+) ion enters excitable cells through voltage-gated calcium channels and is an excellent relaxation agent. In addition, Mn(2+) has been shown to trace neuronal connections in the mouse olfactory and visual systems, enabling MRI neuronal tract tracing. The purpose of the present studies was to determine if these two properties could be combined to trace Mn(2+) from sites of activation in the olfactory epithelium to the olfactory bulb thereby localizing regions within the olfactory bulb that respond to a particular odor. Mice were exposed to an aerosolized solution containing either a high pheromone content odor (male mouse urine) or amyl acetate plus MnCl(2). In both cases the odors caused a localized T(1) MRI enhancement in the olfactory epithelium and bulb that was dependent upon the presence of Mn(2+). The high pheromone containing solution caused enhancement in the anatomically correct location of the accessory olfactory bulb. Amyl acetate also caused T(1)-weighted MRI enhancement in specific regions of the olfactory bulb. These areas showing activation agree well with previous 2-deoxyglucose and BOLD fMRI results in the rat. Using manganese-enhanced MRI (MEMRI) it should be possible to rapidly map a variety of odors. Furthermore, since the effects of activation are imaged after the activation protocol it should be possible to take the time to obtain very high resolution images and make MEMRI maps from awake behaving animals.

Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and cardiomyopathy

Mutations in the cardiac ryanodine receptor 2 (RyR2) have been associated with catecholaminergic polymorphic ventricular tachycardia and a form of arrhythmogenic right ventricular dysplasia. To study the relationship between RyR2 function and these phenotypes, we developed knockin mice with the human disease-associated RyR2 mutation R176Q. Histologic analysis of hearts from RyR2R176Q/+ mice revealed no evidence of fibrofatty infiltration or structural abnormalities characteristic of arrhythmogenic right ventricular dysplasia, but right ventricular end-diastolic volume was decreased in RyR2R176Q/+ mice compared with controls, indicating subtle functional impairment due to the presence of a single mutant allele. Ventricular tachycardia (VT) was observed after caffeine and epinephrine injection in RyR2R176Q/+, but not in WT, mice. Intracardiac electrophysiology studies with programmed stimulation also elicited VT in RyR2R176Q/+ mice. Isoproterenol administration during programmed stimulation increased both the number and duration of VT episodes in RyR2R176Q/+ mice, but not in controls. Isolated cardiomyocytes from RyR2R176Q/+ mice exhibited a higher incidence of spontaneous Ca2+ oscillations in the absence and presence of isoproterenol compared with controls. Our results suggest that the R176Q mutation in RyR2 predisposes the heart to catecholamine-induced oscillatory calcium-release events that trigger a calcium-dependent ventricular arrhythmia.

Statistical diffusion tensor histology reveals regional dysmyelination effects in the shiverer mouse mutant

Shiverer is an important model of central nervous system dysmyelination characterized by a deletion in the gene encoding myelin basic protein with relevance to human dysmyelinating and demyelinating diseases. Perfusion fixed brains from shiverer mutant (C3Fe.SWV Mbp(shi)/Mbp(shi)n = 6) and background control (C3HeB.FeJ, n = 6) mice were compared using contrast enhanced volumetric diffusion tensor magnetic resonance microscopy with a nominal isotropic spatial resolution of 80 mum. Images were accurately coregistered using non-linear warping allowing voxel-wise statistical parametric mapping of tensor invariant differences between control and shiverer groups. Highly significant differences in the tensor trace and both the axial and radial diffusivity were observed within the major white matter tracts and in the thalamus, midbrain, brainstem and cerebellar white matter, consistent with a high density of myelinated axons within these regions. The fractional anisotropy was found to be much less sensitive than the trace and eigenvalues to dysmyelination and associated microanatomic changes.

Overexpression of SOD-2 reduces hippocampal superoxide and prevents memory deficits in a mouse model of Alzheimer's disease.

Alzheimers disease (AD) is a neurodegenerative disease characterized by impaired cognitive function and the deposition of extracellular amyloid plaques and intracellular tangles. Although the proximal cause of AD is not well understood, it is clear that amyloid-β (Aβ) plays a critical role in AD pathology. Recent studies also implicate mitochondrial abnormalities in AD. We investigated this idea by crossing mice that overexpress mitochondrial superoxide dismutase (SOD-2) with the Tg2576 mouse model of AD that overexpresses the human amyloid precursor protein carrying the Swedish mutation (K670N:M671L). We found that overexpression of SOD-2 decreased hippocampal superoxide, prevented AD-related learning and memory deficits, and reduced Aβ plaques. Interestingly, SOD-2 overexpression did not affect the absolute levels of Aβ1–40 and Aβ1–42, but did significantly reduce the Aβ1–42 to Aβ1–40 ratio, thereby shifting the balance toward a less amyloidogenic Aβ composition. These findings directly link mitochondrial superoxide to AD pathology and demonstrate the beneficial effects of a mitochondrial anti-oxidant enzyme, hence offering significant therapeutic implications for AD.

Manganese-enhanced MRI of mouse heart during changes in inotropy†

Recently the dual properties of manganese ion (Mn2+) as an MRI contrast agent and a calcium analogue to enter excitable cells has been used to mark specific cells in brain and as a potential intracellular cardiac contrast agent. Here the hypothesis that in vivo manganese‐enhanced MRI (MEMRI) can detect changes in inotropy in the mouse heart has been tested. T1‐weighted images were acquired every minute during an experimental time course of 75 min. Varying doses of Mn2+ (3.3–14.0 nmoles/min/g BW) were infused during control and altered inotropy with dobutamine (positive inotropy due to increased calcium influx) and the calcium channel blocker diltiazem (negative inotropy). Infusion of MnCl2 led to a significant increase in signal enhancement in mouse heart that saturated above 3.3 ± 0.1 nmoles/min/g BW Mn2+ infusion. At the highest Mn2+ dose infused there was a 41–47% increase in signal intensity with no alteration in cardiac function as measured by MRI‐determined ejection fractions. Dobutamine increased both the steady‐state level of enhancement and the rate of MRI signal enhancement. Diltiazem decreased both the steady‐state level of enhancement and the rate of MRI signal enhancement. These results are consistent with the model that Mn2+‐induced enhancement of cardiac signal is indicative of the rate of calcium influx into the heart. Thus, the simultaneous measurement of global function and calcium influx using MEMRI may provide a useful method of evaluating in vivo responses to inotropic therapy. Magn Reson Med 46:884–890, 2001. Published 2001 Wiley‐Liss, Inc.

In vivo trans-synaptic tract tracing from the murine striatum and amygdala utilizing manganese enhanced MRI (MEMRI)

Small focal injections of manganese ion (Mn2+) deep within the mouse central nervous system combined with in vivo high‐resolution MRI delineate neuronal tracts originating from the site of injection. Previous work has shown that Mn2+ can be taken up through voltage‐gated Ca2+ channels, transported along axons, and across synapses. Moreover, Mn2+ is a paramagnetic MRI contrast agent, causing positive contrast enhancement in tissues where it has accumulated. These combined properties allow for its use as an effective MRI detectable neuronal tract tracer. Injections of low concentrations of MnCl2 into either the striatum or amygdala produced significant contrast enhancement along the known neuronal circuitry. The observed enhancement pattern is different at each injection site and enhancement of the homotopic areas was observed in both cases. Ten days postinjection, the Mn2+ had washed out, as evidenced by the absence of positive contrast enhancement within the brain. This methodology allows imaging of neuronal tracts long after the injection of the ion because Mn2+ concentrates in active neurons and resides for extended periods of time. With appropriate controls, differentiation of subsets of neuronal pathways associated with behavioral and pharmacological paradigms should be feasible. Magn Reson Med 50:33–39, 2003.

Tracking of multimodal therapeutic nanocomplexes targeting breast cancer in vivo.

Nanoparticle-based therapeutics with local delivery and external electromagnetic field modulation holds extraordinary promise for soft-tissue cancers such as breast cancer; however, knowledge of the distribution and fate of nanoparticles in vivo is crucial for clinical translation. Here we demonstrate that multiple diagnostic capabilities can be introduced in photothermal therapeutic nanocomplexes by simultaneously enhancing both near-infrared fluorescence and magnetic resonance imaging (MRI). We track nanocomplexes in vivo, examining the influence of HER2 antibody targeting on nanocomplex distribution over 72 h. This approach provides valuable, detailed information regarding the distribution and fate of complex nanoparticles designed for specific diagnostic and therapeutic functions.

In vivo axonal transport rates decrease in a mouse model of Alzheimer's disease

Axonopathy is a pronounced attribute of many neurodegenerative diseases. In Alzheimers disease (AD), axonal swellings and degeneration are prevalent and may contribute to the symptoms of AD senile dementia. Current limitations in identifying the contribution of axonal damage to AD include the inability to detect when this damage occurs in relation to other identifiers of AD because of the invasiveness of existing methods. To overcome this, we further developed the MRI methodology Manganese Enhanced MRI (MEMRI) to assess in vivo axonal transport rates. Prior to amyloid-beta (Abeta) deposition, the axonal transport rates in the Tg2576 mouse model of AD were normal. As Abeta levels increased and before plaque formation, we observed a significant decrease in axonal transport rates of the Tg2576 mice compared to controls. After plaque formation, the decline in the transport rate in the Tg2576 mice became even more pronounced. These data indicate that in vivo axonal transport rates decrease prior to plaque formation in the Tg2576 mouse model of AD.

Tau Loss Attenuates Neuronal Network Hyperexcitability in Mouse and Drosophila Genetic Models of Epilepsy

Neuronal network hyperexcitability underlies the pathogenesis of seizures and is a component of some degenerative neurological disorders such as Alzheimers disease (AD). Recently, the microtubule-binding protein tau has been implicated in the regulation of network synchronization. Genetic removal of Mapt, the gene encoding tau, in AD models overexpressing amyloid-β (Aβ) decreases hyperexcitability and normalizes the excitation/inhibition imbalance. Whether this effect of tau removal is specific to Aβ mouse models remains to be determined. Here, we examined tau as an excitability modifier in the non-AD nervous system using genetic deletion of tau in mouse and Drosophila models of hyperexcitability. Kcna1−/− mice lack Kv1.1-delayed rectifier currents and exhibit severe spontaneous seizures, early lethality, and megencephaly. Young Kcna1−/− mice retained wild-type levels of Aβ, tau, and tau phospho-Thr231. Decreasing tau in Kcna1−/− mice reduced hyperexcitability and alleviated seizure-related comorbidities. Tau reduction decreased Kcna1−/− video-EEG recorded seizure frequency and duration as well as normalized Kcna1−/− hippocampal network hyperexcitability in vitro. Additionally, tau reduction increased Kcna1−/− survival and prevented megencephaly and hippocampal hypertrophy, as determined by MRI. Bang-sensitive Drosophila mutants display paralysis and seizures in response to mechanical stimulation, providing a complementary excitability assay for epistatic interactions. We found that tau reduction significantly decreased seizure sensitivity in two independent bang-sensitive mutant models, kcc and eas. Our results indicate that tau plays a general role in regulating intrinsic neuronal network hyperexcitability independently of Aβ overexpression and suggest that reducing tau function could be a viable target for therapeutic intervention in seizure disorders and antiepileptogenesis.

Hyperglycemia induces oxidative stress and impairs axonal transport rates in mice.

Background While hyperglycemia-induced oxidative stress damages peripheral neurons, technical limitations have, in part, prevented in vivo studies to determine the effect of hyperglycemia on the neurons in the central nervous system (CNS). While olfactory dysfunction is indicated in diabetes, the effect of hyperglycemia on olfactory receptor neurons (ORNs) remains unknown. In this study, we utilized manganese enhanced MRI (MEMRI) to assess the impact of hyperglycemia on axonal transport rates in ORNs. We hypothesize that (i) hyperglycemia induces oxidative stress and is associated with reduced axonal transport rates in the ORNs and (ii) hyperglycemia-induced oxidative stress activates the p38 MAPK pathway in association with phosphorylation of tau protein leading to the axonal transport deficits. Research Design and Methods T1-weighted MEMRI imaging was used to determine axonal transport rates post-streptozotocin injection in wildtype (WT) and superoxide dismutase 2 (SOD2) overexpressing C57Bl/6 mice. SOD2 overexpression reduces mitochondrial superoxide load. Dihydroethidium staining was used to quantify the reactive oxygen species (ROS), specifically, superoxide (SO). Protein and gene expression levels were determined using western blotting and Q-PCR analysis, respectively. Results STZ-treated WT mice exhibited significantly reduced axonal transport rates and significantly higher levels of ROS, phosphorylated p38 MAPK and tau protein as compared to the WT vehicle treated controls and STZ-treated SOD2 mice. The gene expression levels of p38 MAPK and tau remained unchanged. Conclusion Increased oxidative stress in STZ-treated WT hyperglycemic mice activates the p38 MAPK pathway in association with phosphorylation of tau and attenuates axonal transport rates in the olfactory system. In STZ-treated SOD-overexpressing hyperglycemic mice in which superoxide levels are reduced, these deficits are reversed.