NSF workshop report: Discovering general principles of nervous system organization by comparing brain maps across species

Efforts to understand nervous system structure and function have received new impetus from the federal Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative. Comparative analyses can contribute to this effort by leading to the discovery of general principles of neural circuit design, information processing, and gene‐structure‐function relationships that are not apparent from studies on single species. We here propose to extend the comparative approach to nervous system ‘maps' comprising molecular, anatomical, and physiological data. This research will identify which neural features are likely to generalize across species, and which are unlikely to be broadly conserved. It will also suggest causal relationships between genes, development, adult anatomy, physiology, and, ultimately, behavior. These causal hypotheses can then be tested experimentally. Finally, insights from comparative research can inspire and guide technological development. To promote this research agenda, we recommend that teams of investigators coalesce around specific research questions and select a set of ‘reference species' to anchor their comparative analyses. These reference species should be chosen not just for practical advantages, but also with regard for their phylogenetic position, behavioral repertoire, well‐annotated genome, or other strategic reasons. We envision that the nervous systems of these reference species will be mapped in more detail than those of other species. The collected data may range from the molecular to the behavioral, depending on the research question. To integrate across levels of analysis and across species, standards for data collection, annotation, archiving, and distribution must be developed and respected. To that end, it will help to form networks or consortia of researchers and centers for science, technology, and education that focus on organized data collection, distribution, and training. These activities could be supported, at least in part, through existing mechanisms at NSF, NIH, and other agencies. It will also be important to develop new integrated software and database systems for cross‐species data analyses. Multidisciplinary efforts to develop such analytical tools should be supported financially. Finally, training opportunities should be created to stimulate multidisciplinary, integrative research into brain structure, function, and evolution. J. Comp. Neurol. 522:1445–1453, 2014. © 2014 Wiley Periodicals, Inc.     

Design and characterization of a dynamic vibrational culture system

To engineer a functional vocal fold tissue, the mechanical environment of the native tissue needs to be emulated in vitro. We have created a dynamic culture system capable of generating vibratory stimulations at human phonation frequencies. The novel device is composed of a function generator, a power amplifier, an enclosed loudspeaker and a circumferentially‐anchored silicone membrane. The vibration signals are translated to the membrane aerodynamically by the oscillating air pressure underneath. The vibration profiles detected on the membrane were symmetrical relative to the centre of the membrane as well as the resting position over the range of frequencies (60–300 Hz) and amplitudes tested (1–30 µm). The oscillatory motion of the membrane gave rise to two orthogonal, in‐plane strain components that are similar in magnitude (0.47%) and are strong functions of membrane thickness. Neonatal foreskin fibroblasts (NFFs) attached to the membrane were subjected to a 1 h vibration at 60, 110 and 300 Hz, with the displacement at the centre of the membrane varying in the range 1–30 µm, followed by a 6 h rest. These regimens did not cause morphological changes to the cells. An increase in cell proliferation was detected when NFFs were driven into oscillation at 110 Hz with a normal displacement of 30 µm. qPCR results showed that the expression of genes encoding some extracellular matrix proteins was altered in response to changes in vibratory frequency and amplitude. The dynamic culture device provides a potentially useful in vitro platform for evaluating cellular responses to vibration. Copyright © 2011 John Wiley & Sons, Ltd.     

In vivo 19F magnetic resonance spectroscopy and chemical shift imaging of tri-fluoro-nitroimidazole as a potential hypoxia reporter in solid tumors.

PURPOSE: 2-Nitro-alpha-[(2,2,2-trifluoroethoxy)methyl]-imidazole-1-ethanol (TF-MISO) was investigated as a potential noninvasive marker of tissue oxygen levels in tumors using (19)F magnetic resonance spectroscopy (MRS) and (19)F chemical shift imaging. EXPERIMENTAL DESIGNS: In vitro data were obtained using high-performance liquid chromatography on tumor cells incubated under varying oxygen conditions to determine the oxygen-binding characteristics. In vivo data were obtained using a well-characterized hypoxic murine breast tumor (MCa), in addition to studies on a rat prostate tumor model (R3327-AT) implanted in nude mice. Detection of intratumor (19)F signal from TF-MISO was done using MRS for up to 10 h following a 75 mg/kg i.v. injection. Localized distribution of the compound in the implanted MCa tumor has been imaged using slice-selective two-dimensional chemical shift imaging 6 h after injection. RESULTS: The in vitro results showed that TF-MISO preferentially accumulates in cells incubated under anoxic conditions. The in vivo (19)F MR spectral features (line width and chemical shift) were recorded as a function of time after injection, and the results indicate that the fluorine atoms are indeed sensitive to changes in the local environment while still providing a detectable MR signal. Ex vivo spectra were collected and established the visibility of the (19)F signal under conditions of maximum hypoxia. Late time point (>6 h) tumor tissue concentrations, as obtained from (19)F MRS, suggest that TF-MISO is reduced and retained in hypoxic tumor. The feasibility of obtaining TF-MISO tumor distribution maps in a reasonable time frame was established. CONCLUSIONS: Based on the results presented herein, it is suggested that TF-MISO has the potential to be a valid magnetic resonance hypoxia imaging reporter for both preclinical hypoxia studies and hypoxia-directed clinical therapy.     

Intraaccumbens baclofen selectively enhances feeding behavior in the rat

Intraaccumbens infusions of the GABA(B) agonist baclofen are known to stimulate food intake in the rat. The aim of the present study was to evaluate the effects of baclofen infusion on nonfood-related chewing and on the consumption of a palatable fluid. Rats were bilaterally infused with baclofen (188 ng in 1 microL) or saline, and tested in a situation in which food was available in one or two locations and wood blocks might also be present. Baclofen-infused animals showed no enhancement of chewing directed at the wood blocks, but showed increased food consumption regardless of food location. In a second, separate test we recorded the microstructural parameters for drinking of a palatable glucose/saccharin mixture. Baclofen infusion had no effect on overall intake, although bout size was reduced and the number of bouts was increased. These data confirm that baclofen-stimulated food intake following accumbens infusion is a robust and substantial phenomenon that appears to be selective to solid food. It is likely to result from relatively direct activation of neural circuits for feeding, rather than an indirect facilitation consequent upon changes in taste processings, as has been suggested for some other examples of drug-induced hyperphagia.     

Induction of apoptosis in murine tumors by cyclophosphamide

Whereas there have been several recent reports of the induction of apoptosis by chemotherapy agents in cell culture systems, much less is known about the role of this mode of cell death in tumors treated in vivo. We therefore quantitated the proportion of apoptotic cells induced as a function of time and dose in two murine tumors treated with cyclophosphamide in vivo. The two tumors were a mammary adenocarcinoma, MCa-4, and an ovarian adenocarcinoma, OCa-1. The percent apoptosis was scored from stained histological sections of the tumors using a system based on the characteristic features of the apoptotic nuclei. The kinetics of apoptosis development were determined over a 5-day period following treatment of the mice with 200 mg/kg. The percent apoptosis peaked between 10–18 h in both tumors and then slowly declined to background levels by 5 days after treatment. The dose responses showed that even much lower doses, 25 mg/kg, could induce significant apoptosis and that the proportion of apoptotic cells plateaued at doses higher than 100 mg/kg. These results are compared and contrasted with our previous reports on apoptosis induction in these same tumors with ionizing radiation.The Work reported in this paper was supported by research grants CA-06294 and CA-16672 awarded by the National Cancer Institute, Department of Health and Human Services, and by the Katharine Unsworth Memorial Fund     

Pelvic ultrasound findings in different forms of sexual precocity

Recently produced reference curves for various ultrasound dimensions were used to retrospectively assess 67 pelvic ultrasound scans carried out at the initial presentation in girls with sexual precocity. At presentation the group with precocious puberty had significantly increased uterine lengths and ovarian volumes compared with the normal population, and a significantly increased fundal–cervical ratio. Ovarian volume was also significantly increased in thelarche and thelarche variant. The fundal–cervical ratio was significantly increased in thelarche variant. There was considerable overlap between individuals with sexual precocity and normal subjects. The ultrasound findings that best discriminated early or precocious puberty from other forms of sexual precocity were the presence of a midline endometrial echo, and a uterine length above the 97th centile for age. An entirely normal pelvic ultrasound at presentation did not rule out the possibility of precocious puberty.     

Inhibitory effects of retinoids on development of squamous metaplasia in rat mammary epithelial organoids cultured in Matrigel

Squamous metaplasia (SQM) developed in cultures of rat mammary organoids in reconstituted basement membrane, Matrigel, under either a complete hormone medium (CHM) or a serum-free mammary epithelium growth medium (MEGM). Organoids cultured in CHM gave rise to fewer such SQM (˜5%) than those in MEGM (˜16%). Formation of SQM was completely suppressed when retinoids were added to CHM. However, a few SQM were still observed in cultures in MEGM with added retinoids. Addition of 5% fetal bovine serum suppressed development of SQM cultured in MEGM. Delayed addition of retinoids also inhibited further development of SQM. Development of SQM from mammary epithelial cells is not common, and regulatory molecules other than retinoids apparently are involved in their formation and prevention.