Imaging early stages of the female reproductive structure of arabidopsis by confocal laser scanning microscopy

Background : The gynoecium is the female reproductive structure and probably the most complex plant structure. During its development, different internal tissues and structures are formed. Insights in gene expression or hormone localization patterns are key to understanding gynoecium development from a molecular biology point of view. Results : Imaging with a confocal laser scanning microscope (CLSM) is a widely used strategy; however, visualization of internal developmental expression patterns in the Arabidopsis gynoecium can be technically challenging. Here, we present a detailed protocol that allows the visualization of internal expression patterns at high resolution during gynoecium development. We demonstrate the applicability using a cytokinin response marker (TCS::GFP), an auxin response marker (DR5::VENUS), and a SEPALLATA3 marker (SEP3::SEP3:GFP). Conclusions : The detailed protocol presented here allows the visualization of fluorescence signals in internal structures during Arabidopsis gynoecium development. This protocol may also be adapted for imaging other challenging plant structures or organs. Developmental Dynamics 244:1286–1290, 2015. © 2015 Wiley Periodicals, Inc.     

Perivascular structures in corrosion casts of the human central nervous system: A confocal laser and scanning electron microscope study

Scanning electron microscopy (SEM) of microvascular corrosion casts revealed perivascular structures that resembled smooth muscle and pericyte cells. Although these structures have been studied in widely different experimental contexts, their origin, function, and distribution pattern in different tissues are not understood. Microvascular corrosion casts from 15 fresh human brains and 20 lumbar spinal cords were studied by SEM. In five cerebral hemispheres a fluorescent resin was injected in order to study the vascular bed by confocal laser scanning microscopy (CLSM). Microvascular casts showed two perivascular structures on their surfaces: plastic strips, which formed a muff around arteriolar vessels, and pericyte-like structures that were present around the capillary network. Their morphological characteristics and distribution were similar to those of smooth muscle cells and pericytes, respectively. The SEM study showed that these structures were not tightly joined to the cast surface, but were connected to the vascular cast by narrow plastic connections. The CLSM showed that the resin invaded the subendothelial space, thus giving rise to these structures. Perivascular structures associated with arteriolar and capillary vessels appear to represent smooth muscle cells and pericytes. They are formed by the passage of the resin to the subendothelial space, probably through weak endothelial cell junctions. The effusion of resin into the subendothelial space may represent evidence for the structural basis of myocyte and pericyte cell control. Chemical communication by substances released locally or transported to these cells through these junctions may regulate their functions, allowing them to regulate blood flow. Anat. Rec. 252:176–184, 1998. © 1998 Wiley-Liss, Inc.     

Morphological and biochemical analyses of otoliths of the ice-fish Chionodraco hamatus confirm a common origin with red-blooded species

The morphology and composition of the three otoliths of the Antarctic ice-fish Chionodraco hamatus were studied by scanning electron microscopy and X-ray diffraction. The composition of the sagitta, lapillus and asteriscus protein matrices was also analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, western blots and confocal laser scanning microscopy to reveal the presence of and to localize the calcium-binding proteins calmodulin, calbindin and S-100. Morphological results indicated that the otoliths in this ice-fish were similar to those of Trematomus bernacchii , a red-blooded Antarctic species [B. Avallone et al. (2003 ) J. Submicrosc. Cytol. Pathol. 35 , 69–76], but rather different from those of other teleosts. These two Antarctic species possessed a completely vateritic asteriscus, whereas their sagitta and lapillus were made mostly of aragonite. Parallel analysis of protein patterns in C. hamatus and T. bernacchii revealed that the sagitta significantly differed from the lapillus and asteriscus in both species. The sagitta did not contain the S-100 protein and showed calmodulin and calbindin located in discontinuous or incremental zones, respectively. These results demonstrate that the otoliths of C. hamatus and T. bernacchii share more resemblances than differences and support the idea of a common origin of these species.     

Efferent connections of an auditory area in the caudal insular cortex of the rat: anatomical nodes for cortical streams of auditory processing and cross-modal sensory interactions

In the rat cortex, the two non-primary auditory areas, posterodorsal and ventral auditory areas, may constitute the two streams of auditory processing in their distinct projections to the posterior parietal and insular cortices. The posterior parietal cortex is considered crucial for auditory spatial processing and directed attention, while possible auditory function of the insular cortex is largely unclear. In this study, we electrophysiologically delineated an auditory area in the caudal part of the granular insular cortex (insular auditory area, IA) and examined efferent connections of IA with anterograde tracer biocytin to deduce the functional significance of IA. IA projected to the rostral agranular insular cortex, a component of the lateral prefrontal cortex. IA also projected to the adjacent dysgranular insular cortex and the caudal agranular insular cortex and sent feedback projections to cortical layer I of the primary and secondary somatosensory areas. Corticofugal projections terminated in auditory, somatosensory and visceral thalamic nuclei, and the bottom of the thalamic reticular nucleus that could overlap the visceral sector. The ventral part of the caudate putamen, the external cortex of the inferior colliculus and the central amygdaloid nucleus were also the main targets. IA exhibited neural response to transcutaneous electrical stimulation of the forepaw in addition to acoustic stimulation (noise bursts and pure tones). The results suggest that IA subserves diverse functions associated with somatosensory, nociceptive and visceral processing that may underlie sound-driven emotional and autonomic responses. IA, being potentially involved in such extensive cross-modal sensory interactions, could also be an important anatomical node of auditory processing linked to higher neural processing in the prefrontal cortex.     

Electron Microscopic and Confocal Laser Scanning Microscopic Observation of Subcellular Organelles and Pituitary Hormone mRNA: Application of Ultrastructural In Situ Hybridization and Immunohistochemistry to the Pathophysiological Studies of Pituitary Cells

Nonradioisotopic electron microscopic (EM)in situ hybridization (ISH) (EM-ISH) with biotinylated oligonucleotide probes is utilized for the ultrastructural visualization of pituitary hormone mRNA in rat pituitary cells. EM-ISH is an important tool for clarifying the intracellular localization of mRNA and the exact site of specific hormone synthesis on the rough endoplasmic reticulum. The simultaneous visualization of mRNA and encoded protein in the same cell using preembedding EM-ISH and subsequent postembedding immunoreaction with protein A colloidal gold complex can provide an important clue for elucidating the intracellular correlation of mRNA translation and secretion of translated protein. Another focus of this review is the utilization of a recently developed imaging system of confocal laser scanning microscopy (CLSM). The combination of CLSM and image analysis system (IAS) enables us to visualize an individual dimensional image of the intracellular distribution of mRNA and subcellular organelles successfully at any optional cross sections of light microscopic ISH studies, and can be another useful tool for the ultrastructural ISH study of mRNA.     

“The developmental and functional logic of neuronal circuits”: commentary on the Kavli Prize in Neuroscience

The first Kavli Prize in Neuroscience recognizes a confluence of career achievements that together provide a fundamental understanding of how brain and spinal cord circuits are assembled during development and function in the adult. The members of the Kavli Neuroscience Prize Committee have decided to reward three scientists (Sten Grillner, Thomas Jessell, and Pasko Rakic) jointly “for discoveries on the developmental and functional logic of neuronal circuits”.