Faculty Bibliography

We have investigated the source(s) and targeting of components to PNS nodes of Ranvier. We show adhesion molecules are freely diffusible within the axon membrane and accumulate at forming nodes from local sources, whereas ion channels and cytoskeletal components are largely immobile and require transport to the node. We further characterize targeting of NF186, an adhesion molecule that pioneers node formation. NF186 redistributes to nascent nodes from a mobile, surface pool. Its initial accumulation and clearance from the internode require extracellular interactions, whereas targeting to mature nodes, i.e., those flanked by paranodal junctions, requires intracellular interactions. After incorporation into the node, NF186 is immobile, stable, and promotes node integrity. Thus, nodes assemble from two sources: adhesion molecules, which initiate assembly, accumulate by diffusion trapping via interactions with Schwann cells, whereas ion channels and cytoskeletal components accumulate via subsequent transport. In mature nodes, components turnover slowly and are replenished via transport. VIDEO ABSTRACT:

Neuronal oscillations allow for temporal segmentation of neuronal spikes. Interdependent oscillators can integrate multiple layers of information. We examined phase-phase coupling of theta and gamma oscillators in the CA1 region of rat hippocampus during maze exploration and rapid eye movement sleep. Hippocampal theta waves were asymmetric, and estimation of the spatial position of the animal was improved by identifying the waveform-based phase of spiking, compared to traditional methods used for phase estimation. Using the waveform-based theta phase, three distinct gamma bands were identified: slow gamma(S) (gamma(S); 30-50 Hz), midfrequency gamma(M) (gamma(M); 50-90 Hz), and fast gamma(F) (gamma(F); 90-150 Hz or epsilon band). The amplitude of each sub-band was modulated by the theta phase. In addition, we found reliable phase-phase coupling between theta and both gamma(S) and gamma(M) but not gamma(F) oscillators. We suggest that cross-frequency phase coupling can support multiple time-scale control of neuronal spikes within and across structures.

Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with light-sensitive ligands, innately 'blind' neurobiological receptors can be converted into photoreceptors, which allows them to be photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design, we produced heteromeric alpha3beta4 and alpha4beta2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric ligand-gated ion channels.

Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas sp. The DNA-binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within 1 week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using quantitative reverse-transcription PCR and Surveyor nuclease, respectively. The TALE toolbox described here will enable a broad range of biological applications.

The development of rapid volumetric imaging systems for functional multiphoton microscopy is essential for dynamical imaging of large-scale neuronal network activity. Here, we introduce a line-illuminating temporal-focusing microscope capable of rapid three-dimensional imaging at 10-20 volumes/sec, and study the system's characteristics both theoretically and experimentally. We demonstrate that our system is capable of functional volumetric calcium imaging of distributed neuronal activity patterns, and introduce a computational strategy for activity reconstruction in strongly scattering media.

To evaluate molecular signatures of an individual cell type in comparison to the associated region relevant towards understanding the pathogenesis of Alzheimer's disease (AD), CA1 pyramidal neurons and the surrounding hippocampal formation were microaspirated via laser capture microdissection (LCM) from neuropathologically confirmed AD and age-matched control (CTR) subjects as well as from wild type mouse brain using single population RNA amplification methodology coupled with custom-designed microarray analysis with real-time quantitative polymerase-chain reaction (qPCR) validation. CA1 pyramidal neurons predominantly displayed downregulation of classes of transcripts related to synaptic transmission in AD versus CTR. Regional hippocampal dissections displayed downregulation of several overlapping genes found in the CA1 neuronal population related to neuronal expression, as well as upregulation of select transcripts indicative of admixed cell types including glial-associated markers and immediate-early and cell death genes. Gene level distributions observed in CA1 neurons and regional hippocampal dissections in wild type mice paralleled expression mosaics seen in postmortem human tissue. Microarray analysis was validated in qPCR studies using human postmortem brain tissue and CA1 sector and regional hippocampal dissections obtained from a mouse model of AD/Down syndrome (Ts65Dn mice) and normal disomic (2N) littermates. Classes of transcripts that have a greater percentage of the overall hybridization signal intensity within single neurons tended to be genes related to neuronal communication. The converse was also found, as classes of transcripts such as glial-associated markers were under represented in CA1 pyramidal neuron expression profiles relative to regional hippocampal dissections. These observations highlight a dilution effect that is likely to occur in conventional regional microarray and qPCR studies. Thus, single population studies of specific neurons and intrinsic circuits will likely yield informative gene expression profile data that may be subthreshold and/or underrepresented in regional studies with an admixture of cell types

Background: The ability to predict the course of multiple sclerosis (MS) is highly desirable but lacking. Objective: To test whether the MS Severity Scale (MSSS) and global neuronal viability, assessed through the quantification of the whole-brain N-acetylaspartate concentration (WBNAA), concur or complement the assessment of individual patients' disease course. Methods: The MSSS and average WBNAA loss rate (DeltaWBNAA, extrapolated based on one current measurement and the assumption that at disease onset neural sparing was similar to healthy controls, obtained with proton magnetic resonance (MR) spectroscopy and magnetic resonance imaging (MRI)) from 61 patients with MS (18 male and 43 female) with long disease duration (15 years or more) were retrospectively examined. Some 27 patients exhibited a 'benign' disease course, characterized by an Expanded Disability Status Scale score (EDSS) of 3.0 or less, and 34 were 'non-benign': EDSS score higher than 3.0. Results: The two cohorts were indistinguishable in age and disease duration. Benign patients' EDSS and MSSS (2.1 +/- 0.7, 1.15 +/- 0.60) were significantly lower than non-benign (4.6 +/- 1.0, 3.6 +/- 1.2; both p < 10(-4)). Their respective average DeltaWBNAA, 0.10 +/- 0.16 and 0.11 +/- 0.12 mM/year, however, were not significantly different (p > 0.7). While MSSS is both sensitive to (92.6%) and specific for (97.0%) benign MS, DeltaWBNAA is only sensitive (92.6%) but not specific (2.9%). Conclusion: Since the WBNAA loss rate is similar in both phenotypes, the only difference between them is their clinical classification, characterized by MSSS and EDSS. This may indicate that 'benign' MS probably reflects fortuitous sparing of clinically eloquent brain regions and better utilization of brain plasticity

OBJECT: Recent technological developments open the field of therapeutic application of focused ultrasound to the brain through the intact cranium. The goal of this study was to apply the new transcranial magnetic resonance imaging-guided focused ultrasound (tcMRgFUS) technology to perform noninvasive central lateral thalamotomies (CLTs) as a treatment for chronic neuropathic pain. METHODS: In 12 patients suffering from chronic therapy-resistant neuropathic pain, tcMRgFUS CLT was proposed. In 11 patients, precisely localized thermal ablations of 3-4 mm in diameter were produced in the posterior part of the central lateral thalamic nucleus at peak temperatures between 51 degrees C and 64 degrees C with the aid of real-time patient monitoring and MR imaging and MR thermometry guidance. The treated neuropathic pain syndromes had peripheral (5 patients) or central (6 patients) origins and covered all body parts (face, arm, leg, trunk, and hemibody). RESULTS: Patients experienced mean pain relief of 49% at the 3-month follow-up (9 patients) and 57% at the 1-year follow-up (8 patients). Mean improvement according to the visual analog scale amounted to 42% at 3 months and 41% at 1 year. Six patients experienced immediate and persisting somatosensory improvements. Somatosensory and vestibular clinical manifestations were always observed during sonication time because of ultrasound-based neuronal activation and/or initial therapeutic effects. Quantitative electroencephalography (EEG) showed a significant reduction in EEG spectral overactivities. Thermal ablation sites showed sharply delineated ellipsoidal thermolesions surrounded by short-lived vasogenic edema. Lesion reconstructions (18 lesions in 9 patients) demonstrated targeting precision within a millimeter for all 3 coordinates. There was 1 complication, a bleed in the target with ischemia in the motor thalamus, which led to the introduction of 2 safety measures, that is, the detection of a potential cavitation by a cavitation detector and the maintenance of sonication temperatures below 60 degrees C. CONCLUSIONS: The authors assert that tcMRgFUS represents a noninvasive, precise, and radiation-free neurosurgical technique for the treatment of neuropathic pain. The procedure avoids mechanical brain tissue shift and eliminates the risk of infection. The possibility of applying sonication thermal spots free from trajectory restrictions should allow one to optimize target coverage. The real-time continuous MR imaging and MR thermometry monitoring of targeting accuracy and thermal effects are major factors in optimizing precision, safety, and efficacy in an outpatient context.