A Comprehensive Integrated Anatomical and Molecular Atlas of Rat Intrinsic Cardiac Nervous System.

We have developed and integrated several technologies including whole-organ imaging and software development to support an initial precise 3D neuroanatomical mapping and molecular phenotyping of the intracardiac nervous system (ICN). While qualitative and gross anatomical descriptions of the anatomy of the ICN have each been pursued, we here bring forth a comprehensive atlas of the entire rat ICN at single-cell resolution. Our work precisely integrates anatomical and molecular data in the 3D digitally reconstructed whole heart with resolution at the micron scale. We now display the full extent and the position of neuronal clusters on the base and posterior left atrium of the rat heart, and the distribution of molecular phenotypes that are defined along the base-to-apex axis, which had not been previously described. The development of these approaches needed for this work has produced method pipelines that provide the means for mapping other organs.

Three-dimensional Imaging and Scanning: Current and Future Applications for Pathology.

Imaging is vital for the assessment of physiologic and phenotypic details. In the past, biomedical imaging was heavily reliant on analog, low-throughput methods, which would produce two-dimensional images. However, newer, digital, and high-throughput three-dimensional (3D) imaging methods, which rely on computer vision and computer graphics, are transforming the way biomedical professionals practice. 3D imaging has been useful in diagnostic, prognostic, and therapeutic decision-making for the medical and biomedical professions. Herein, we summarize current imaging methods that enable optimal 3D histopathologic reconstruction: Scanning, 3D scanning, and whole slide imaging. Briefly mentioned are emerging platforms, which combine robotics, sectioning, and imaging in their pursuit to digitize and automate the entire microscopy workflow. Finally, both current and emerging 3D imaging methods are discussed in relation to current and future applications within the context of pathology.

Specimen preparation, imaging, and analysis protocols for knife-edge scanning microscopy.

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM), developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi), vascular networks (India ink), and cell body distribution (Nissl). The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

Multiscale exploration of mouse brain microstructures using the knife-edge scanning microscope brain atlas.

Connectomics is the study of the full connection matrix of the brain. Recent advances in high-throughput, high-resolution 3D microscopy methods have enabled the imaging of whole small animal brains at a sub-micrometer resolution, potentially opening the road to full-blown connectomics research. One of the first such instruments to achieve whole-brain-scale imaging at sub-micrometer resolution is the Knife-Edge Scanning Microscope (KESM). KESM whole-brain data sets now include Golgi (neuronal circuits), Nissl (soma distribution), and India ink (vascular networks). KESM data can contribute greatly to connectomics research, since they fill the gap between lower resolution, large volume imaging methods (such as diffusion MRI) and higher resolution, small volume methods (e.g., serial sectioning electron microscopy). Furthermore, KESM data are by their nature multiscale, ranging from the subcellular to the whole organ scale. Due to this, visualization alone is a huge challenge, before we even start worrying about quantitative connectivity analysis. To solve this issue, we developed a web-based neuroinformatics framework for efficient visualization and analysis of the multiscale KESM data sets. In this paper, we will first provide an overview of KESM, then discuss in detail the KESM data sets and the web-based neuroinformatics framework, which is called the KESM brain atlas (KESMBA). Finally, we will discuss the relevance of the KESMBA to connectomics research, and identify challenges and future directions.

Telemedicine for audiology screening of infants.

Distortion product otoacoustic emissions (DPOAE) and automated auditory brainstem response (AABR) screening were conducted in infants at a distant hospital using remote computing. Eighteen males and twelve females ranging in age from 11-45 days were tested. Both DPOAE and AABR data were recorded using an integrated test system which was connected to the computer network at the Utah Valley Regional Medical Center. Using a broadband Internet connection, an examiner at Utah State University, 200 km away, could control the DPOAE and the ABR equipment. Identical hearing screening results were obtained for face-to-face and telemedicine trials with all infants. The DPOAE means for face-to-face and telemedicine trials were not significantly different at any frequency. In an analysis of variance, there was no significant difference for the test method (F = 0.8, P > 0.05). These results indicate that remote computing is a feasible telemedicine method for providing DPOAE and ABR hearing screening services to infants in rural communities.

Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.

Brain, liver, kidney, heart, and skeletal muscle from fatty liver dystrophy (fld/fld) mice, which do not express lipin 1 (lipin), contained much less Mg(2+)-dependent phosphatidic acid phosphatase (PAP) activity than tissues from wild type mice. Lipin harboring the fld(2j) (Gly(84) --> Arg) mutation exhibited relatively little PAP activity. These results indicate that lipin is a major PAP in vivo and that the loss of PAP activity contributes to the fld phenotype. PAP activity was readily detected in immune complexes of lipin from 3T3-L1 adipocytes, where the protein was found both as a microsomal form and a soluble, more highly phosphorylated, form. Fifteen phosphorylation sites were identified by mass spectrometric analyses. Insulin increased the phosphorylation of multiple sites and promoted a gel shift that was due in part to phosphorylation of Ser(106). In contrast, epinephrine and oleic acid promoted dephosphorylation of lipin. The PAP-specific activity of lipin was not affected by the hormones or by dephosphorylation of lipin with protein phosphatase 1. However, the ratio of soluble to microsomal lipin was markedly increased in response to insulin and decreased in response to epinephrine and oleic acid. The results suggest that insulin and epinephrine control lipin primarily by changing localization rather than intrinsic PAP activity.

The arrest of biological time as a bridge to engineered negligible senescence.

Biological systems can remain unchanged for several hundred years at cryogenic temperatures. In several hundred years, current rapid scientific and technical progress should lead to the ability to reverse any biological damage whose reversal is not forbidden by physical law. We therefore explore whether contemporary people facing terminal conditions might be preserved well enough today for their eventual recovery to be compatible with physical law. The ultrastructure of the brain can now be excellently preserved by vitrification, and solutions needed for vitrification can now be distributed through organs with retention of organ viability after transplantation. Current law requires a few minutes of cardiac arrest before cryopreservation of terminal patients, but dogs and cats have recovered excellent brain function after 16-60 min of complete cerebral ischemia. The arrest of biological time as a bridge to engineered negligible senescence, therefore, appears consistent with current scientific and medical knowledge.

Selective attention affects human brain stem frequency-following response.

Selective attention modifies long-latency cortical event-related potentials. Amplitudes are typically enhanced and/or latencies reduced when evoking stimuli are attended. However, there is controversy concerning the effects of selective attention on short-latency brain stem evoked potentials. The objective of the present study was to assess possible attention effects on the brain stem auditory frequency-following response (FFR) elicited by a periodic tone. Young adult subjects heard a repetitive auditory stimulus while detecting infrequent target stimuli in either an auditory or visual detection task. Five channels of high frequency electroencephalographic (EEG) activity were recorded along the scalp midline with the center electrode positioned at the vertex. The FFR was elicited by the repetitive tone during both tasks. There were significant individual differences in the electrode sites yielding maximum response amplitudes, but overall FFR amplitudes were significantly larger during the auditory attention task. These results suggest that selective attention in humans can modify signal processing in sensory (afferent) pathways at the level of the brain stem. This may reflect top-down perceptual preprocessing mediated by extensive descending (efferent) pathways that originate in the cortex. Overall, the FFR appears to be a robust indicator of early auditory neural processing and shows effects not seen in brain stem auditory evoked response studies employing transient (click) acoustic stimuli.

Insulin-stimulated phosphorylation of lipin mediated by the mammalian target of rapamycin.

The phosphorylation of a previously uncharacterized protein of apparent M(r) approximately 140,000 was found to be increased when rat adipocytes were incubated with insulin. The sequences of peptides generated by digesting the protein with trypsin matched perfectly with sequences in mouse lipin. Lipin is the product of the gene that is mutated in fatty liver dystrophy (fld) mice [Peterfy, M., Phan, J., Xu, P. & Reue, K (2001) Nat. Genet. 27, 121-124], which exhibit several phenotypic abnormalities including hyperlipidemia, defects in adipocyte differentiation, impaired glucose tolerance, and slow growth. When immunoblots were prepared with lipin antibodies, both endogenous adipocyte lipin and recombinant lipin overexpressed in HEK293 cells appeared as bands ranging in apparent M(r) from 120,000 to 140,000. Incubating adipocytes with insulin decreased the electrophoretic mobility and stimulated the phosphorylation of both Ser and Thr residues in lipin. The effects of insulin were abolished by inhibitors of phosphatidylinositol 3-OH kinase, and by rapamycin, a specific inhibitor of the mammalian target of rapamcyin (mTOR). The inhibition by rapamycin was blocked by FK506, which competitively inhibits those effects of rapamycin that are mediated by inhibition of mTOR. Moreover, amino acids, which activate mTOR, mimicked insulin by increasing lipin phosphorylation in a rapamycin-sensitive manner. Thus, lipin represents a target of the mTOR pathway, and potentially links this nutrient-sensing pathway to adipocyte development.