Optimized Protocol for Imaging Cleared Neural Tissues Using Light Microscopy

Understanding physical and chemical processes at an organismal scale is a fundamental goal in biology. While science is adept at explaining biological phenomena at both molecular and cellular levels, understanding how these processes translate to organismal functions remains a challenging problem. This issue is particularly significant for the nervous system where cell signaling and synaptic activities function in the context of broad neural networks. Recent progress in tissue clearing technologies lessens the barriers that previously prevented the study of large tissue samples while maintaining molecular and cellular resolution. While these new methods open vast opportunities and exciting new questions, the logistics of analyzing cellular processes in intact tissue have to be carefully considered. In this protocol, we outline a procedure to rapidly image intact brain tissue up to thousands of cubic millimeters. This experimental pipeline involves three steps: tissue clearing, tissue imaging, and data analysis. In an attempt to streamline the process for researchers entering this field, we address important considerations for each of these stages and describe an integrated solution to image intact biological tissues. Hopefully, this optimized protocol will lower the barrier of implementing high-resolution tissue imaging and facilitate the investigations of mesoscale questions at molecular and cellular resolution

Imagerie dans le cathétérisme des cardiopathies congénitales : place de l’échocardiographie 3D transthoracique

RésuméL’échocardiographie 3D transthoracique a longtemps été freinée dans son développement en raison de conditions techniques d’acquisition compliquées et de qualité d’images médiocres. L’avènement des sondes matricielles permet au 3D en devenant temps réel d’entrer dans la pratique clinique courante. Si la voie œsophagienne a permis au 3D de trouver ses lettres de noblesses par ses descriptions anatomiques uniques des valves et des septa, l’échocardiographie transthoracique peut désormais se décliner en modes 2D, Doppler et 3D. Ses applications dans la cardiologie congénitale et pédiatrique sont multiples : description anatomique précise des défauts septaux auriculaires et ventriculaires, classification des bicuspidies aortiques et analyse du mécanisme de sténose. Ainsi, l’échocardiographie 3D permet-elle de sélectionner de façon non invasive les patients, de guider et de juger du résultat d’un cathétérisme interventionnel. L’imagerie 3D est un excellent moyen de communication entre l’imageur et le cardiologue interventionnel mais aussi de délivrer des informations claires au patient et à la famille avant et après un cathétérisme.SummaryThree-dimensional echocardiography has improved dramatically due to technical advances in probe design and computer processing. The introduction of real time 3D echocardiography has led to its use in everyday clinical practice. Congenital heart disease demands a detailed understanding of the spatial relationships of cardiac structures to plan treatment. The introduction of new transthoracic 3D probes has extended the applications to real-time guidance of catheter procedures. Prominent among the cardiac lesions which have been studied are: atrial septal defects, ventricular septal defects and stenotic bicuspid aortic valves. Its values should be decisive in many congenital cardiac lesions requiring interventional catheterisation. 3D echocardiography is an easy way to communicate to the patient and its family about the pathology

Methods Of Gene Expression Monitoring

The invention provides methods of monitoring expression of a plurality of genes in a cell or small population of cells. Preferred methods entail contacting an array of probes with a population of nucleic acids derived from a population of fewer than 1000 cells then determining the relative hybridization of the probes to the population of nucleic acid as a measure of the relative representation of genes from the cells. The invention further provides methods of classifying cells. These preferred methods entail determining an expression profile of each of a plurality of cells then classifying the cells in clusters determined by similarity of expression profile. The invention further provides methods of monitoring differentiation of a cell lineage. These preferred methods entail determining an expression profile of each of a plurality of cells at different differentiation stages within the lineage. These cells can then be classified into clusters determined by similarity of expression profile. The clusters can then be ordered by similarity of expression profile. A time course of expression levels for each of the plurality of genes at different stages of differentiation in the cell lineage can then be determined

More Functional V1R Genes Occur in Nest-Living and Nocturnal Terricolous Mammals

Size of the vomeronasal type 1 receptor (V1R) gene repertoire may be a good indicator for examining the relationship between animal genomes and their environmental niche specialization, especially the relationship between ecological factors and the molecular evolutionary history of the sensory system. Recently, Young et al. (Young JM, Massa HF, Hsu L, Trask BJ. 2009. Extreme variability among mammalian V1R gene families. Genome Res.) concluded that no single ecological factor could explain the extreme variability of the V1R gene repertoire in mammalian genomes. In contrast, we found a significant positive correlation between the size and percentage of intact V1R genes in 32 species that represent the phylogenetic diversity of terricolous mammals and two ecological factors: spatial activity and rhythm activity. Nest-living species possessed a greater number of intact V1R genes than open-living species, and nocturnal terricolous mammals tended to possess more intact V1R genes than did diurnal species. Moreover, our analysis reveals that the evolutionary mechanisms underlying these observations likely resulted from the rapid gene birth and accelerated amino acid substitutions in nest-living and nocturnal mammals, likely a functional requirement for exploiting narrow, dark environments. Taken together, these results reveal how adaptation to divergent circadian rhythms and spatial activity were manifested at the genomic scale. Size of the V1R gene family might have indicated how this gene family adapts to ecological factors

A Behavioral Odor Similarity “Space” in Larval Drosophila

To provide a behavior-based estimate of odor similarity in larval Drosophila, we use 4 recognition-type experiments: 1) We train larvae to associate an odor with food and then test whether they would regard another odor as the same as the trained one. 2) We train larvae to associate an odor with food and test whether they prefer the trained odor against a novel nontrained one. 3) We train larvae differentially to associate one odor with food, but not the other one, and test whether they prefer the rewarded against the nonrewarded odor. 4) In an experiment like (3), we test the larvae after a 30-min break. This yields a combined task-independent estimate of perceived difference between odor pairs. Comparing these perceived differences to published measures of physicochemical difference reveals a weak correlation. A notable exception are 3-octanol and benzaldehyde, which are distinct in published accounts of chemical similarity and in terms of their published sensory representation but nevertheless are consistently regarded as the most similar of the 10 odor pairs employed. It thus appears as if at least some aspects of olfactory perception are “computed” in postreceptor circuits on the basis of sensory signals rather than being immediately given by them

Common Promoter Elements in Odorant and Vomeronasal Receptor Genes

In mammals, odorants and pheromones are detected by hundreds of odorant receptors (ORs) and vomeronasal receptors (V1Rs and V2Rs) expressed by sensory neurons that are respectively located in the main olfactory epithelium and in the vomeronasal organ. Even though these two olfactory systems are functionally and anatomically separate, their sensory neurons show a common mechanism of receptor gene regulation: each neuron expresses a single receptor gene from a single allele. The mechanisms underlying OR and VR gene expression remain unclear. Here we investigated if OR and V1R genes share common sequences in their promoter regions