Daniel M. Sforza

Mitochondrial Dysfunction, Oxidative Stress, and Apoptosis Revealed by Proteomic and Transcriptomic Analyses of the Striata in Two Mouse Models of Parkinson’s Disease

The molecular mechanisms underlying the changes in the nigrostriatal pathway in Parkinsons disease (PD) are not completely understood. Here, we use mass spectrometry and microarrays to study the proteomic and transcriptomic changes in the striatum of two mouse models of PD, induced by the distinct neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH). Proteomic analyses resulted in the identification and relative quantification of 912 proteins with two or more unique peptides and 86 proteins with significant abundance changes following neurotoxin treatment. Similarly, microarray analyses revealed 181 genes with significant changes in mRNA, following neurotoxin treatment. The combined protein and gene list provides a clearer picture of the potential mechanisms underlying neurodegeneration observed in PD. Functional analysis of this combined list revealed a number of significant categories, including mitochondrial dysfunction, oxidative stress response, and apoptosis. These results constitute one of the largest descriptive data sets integrating protein and transcript changes for these neurotoxin models with many similar end point phenotypes but distinct mechanisms.

High-resolution voxelation mapping of human and rodent brain gene expression

Voxelation allows high-throughput acquisition of multiple volumetric images of brain gene expression, similar to those obtained from biomedical imaging systems. To obtain these images, the method employs analysis of spatially registered voxels (cubes). For creation of high-resolution maps using voxelation, relatively small voxel sizes are necessary and instruments will be required for semiautomated harvesting of such voxels. Here, we describe two devices that allow spatially registered harvesting of voxels from the human and rodent brain, giving linear resolutions of 3.3 and 1 mm, respectively. Gene expression patterns obtained using these devices showed good agreement with known expression patterns. The voxelation instruments and their future iterations represent a valuable approach to the genome scale acquisition of gene expression patterns in the human and rodent brain.

Postmortem high-resolution 3-dimensional imaging of the primate brain: blockface imaging of perfusion stained tissue.

Modern anatomy of the brain is primarily concerned with visualizing contrast between tissue elements and with 3-dimensional (3-D) reconstruction. The first objective relies on specific neuroanatomical and imaging protocols, the latter on proper alignment of serial cross-sectional anatomy. We present a method that meets these goals at a resolution that allows for the structural delineation in the primate central nervous system. Methylene blue was administered by perfusion to the intact brain of three rhesus macaques thereby staining the neuropil in the cortex and gray nuclei of the brain in situ. Subsequent dissection and serial sectioning revealed excellent contrast in the tissue and showed clear boundaries between anatomical structures. We captured sequential images of the block surface of the brain before each serial section was cut from the specimen by means of a digital camera mounted directly over the microtome stage and the brain. We developed a suite of algorithms to automate the alignment, segmentation, normalization, and intensity correction for the series of blockface images. These digital images were the basis for 3-D reconstruction of the brains.

Gene expression is differentially regulated by neurotransmitters in embryonic neuronal cortical culture

Neurotransmitters and their receptors have been involved in both proper brain development and neurodevelopmental disorders. The role that nicotinic receptors play in immature cortical neurons was initially investigated by gene profiling using Affymetrix DNA arrays. Both short (15 min) and prolonged (18 h) treatments with nicotine did not induce modification in gene expression, whereas a significant down‐regulation of c‐fos protein levels was observed after 18 h treatment. Conversely, a brief treatment with the glutamatergic agonist NMDA triggered up‐regulation of immediate early genes and transcription factors, which remained unaffected by pre‐treatment for 18 h with nicotine. Calcium imaging studies revealed that NMDA activated a sustained increase in intracellular calcium concentration in the majority of neurons, whereas nicotine evoked only a transient calcium increase in a smaller percentage of neurons, suggesting that the calcium signalling response was correlated with activation of gene expression. Nicotine effects on immature cortical neurons perhaps do not require gene regulation but may be still acting on signalling pathways.

Genetic and Genomic Strategies in Learning and Memory

Learning and memory is a property of central importance in the nervous system, yet many of the molecular mechanisms for this behavior remain enshrouded in mystery. Despite the daunting nature of the problem, a number of complementary strategies have been employed to unravel the complexities of learning and memory, ranging from genetics to biochemistry. One of the most recent tools brought to bear in this area is genomics. Here, we review some of the most significant insights that have been so far obtained in learning and memory, and we suggest possible areas of future progress.

Voxelation Methods for Genome Scale Imaging of Brain Gene Expression

Publisher Summary This chapter describes the various aspects of voxelation methods for genome scale imaging of the brain gene expression. Imaging techniques have granted access to significant results on structure and function in the central nervous system. In addition, the completion of the human genome sequence promises to accelerate even further the developments of molecular biology. Voxelation has permitted high-throughput gene expression reconstructions in the human and rodent brains. One study using human brains compared gene expression between the brains from normal individuals and the brains from individuals with Alzheimers disease. Proper use of the described fixation and cryoprotection protocol allows the use of histologic stains to improve contrast in the voxelated section and facilitate image registration. It is important to obtain digital pictures of the selected slab before and after voxelation in order to evaluate possible deformation induced by the procedure. Voxelation studies yield large amounts of data, and a variety of different analytical techniques can be employed for data mining. High-resolution voxelation can result in demanding data sets for image reconstruction. The manual selection of corresponding fiducial landmarks on images allows the algorithm to warp the desired image onto the atlas reference. It is found that gene expression images are further improved using interpolation and smoothing functions.

Anatomical Methods for Voxelation of the Mammalian Brain

Voxelation allows high-throughput acquisition of three-dimensional gene expression patterns in the brain through analysis of spatially registered voxels (cubes). The method results in multiple volumetric maps of gene expression analogous to the images reconstructed in biomedical imaging techniques. An important issue for voxelation is the development of approaches to anchor correctly harvested voxels to the underlying anatomy. Here, we describe experiments to identify fixation and cryopreservation protocols for improved registration of harvested voxels with neuroanatomical structures. Paraformaldehyde fixation greatly reduced RNA recovery as judged by ribosomal RNA abundance. However, gene expression signals from paraformaldehyde-fixed samples were not appreciably diminished as judged by average signal–noise ratios from microarrays, highlighting the difficulties of accurate quantitation of cross-linked RNA. Additional use of cryoprotection helped to improve further RNA recovery and signal from fixed tissue. It appears that the best protocol to provide the necessary resolution of neuroanatomical information in voxelation entails a controlled dose of fixation and thorough cryoprotection, complemented by histological staining.

Operator ordering for generally covariant systems

Abstract The constraint operators belonging to a generally covariant system are found out within the framework of the BRST formalism. The result embraces quadratic Hamiltonian constraints whose potential can be factorized as a never null function times a gauge invariant function. The building of the inner product between physical states is analyzed for systems featuring either intrinsic or extrinsic time.

BRST operator quantization of generally covariant gauge systems

The BRST generator is realized as a Hermitian nilpotent operator for a finite-dimensional gauge system featuring a quadratic super-Hamiltonian and linear supermomentum constraints. As a result, the emerging ordering for the Hamiltonian constraint is not trivial, because the potential must enter the kinetic term in order to obtain a quantization invariant under scaling. Namely, BRST quantization does not lead to the curvature term used in the literature as a means to get that invariance. The inclusion of the potential in the kinetic term, far from being unnatural, is beautifully justified in light of the Jacobi`s principle. {copyright} {ital 1997} {ital The American Physical Society}