We investigated the responses of 114 cells in the barrel cortex of rats to describe the temporal characteristics of excitatory interactions among neurons serving two vibrissae. To examine these interactions, the principal whisker and one adjacent whisker in the same row were stimulated simultaneously or serially at various interstimulus intervals (ISIs). In 37% of the cells tested, combined stimulation of two whiskers exhibited response facilitation; the response to the combined stimulus was larger than the sum of the responses to stimulation of the individual whiskers. The occurrence and magnitude of the facilitation were strongly dependent on the ISI. The ISI capable of producing facilitation for a particular cell was tuned to a narrow range (mean +/- SD, 5.3 +/- 2.3 msec). The ISI that evoked the maximal facilitation was 1.3 +/- 1.3, 3.4 +/- 2.3, and 2.8 +/- 4.5 msec for neurons in layers II/III, IV, and V/VI, respectively. These ISIs corresponded to the difference in latencies between the responses to the individual stimulations of the principal and adjacent whiskers. A significant response facilitation was observed in the regular-spiking cells but not in the fast-spiking cells. When the ISI was longer than the range that evoked facilitation, a suppression of the response to the second whisker stimulation was observed. Facilitation was observed predominantly in layer II/III cells (69%) and to a lesser extent in cells of layers IV (15%) and V/VI (24%). Our results suggest that, in the barrel cortex, the temporal relationships among tactile stimuli are coded by facilitatory and inhibitory interactions among neurons located in neighboring barrel columns.

To understand the physiological properties and anatomical organization of the spatiotemporal interaction of the responses to multiwhisker stimulation in neurons of the rat barrel cortex, single-unit recordings of 114 neurons were performed across all layers (layer II/III, n ϭ 39; IV, n ϭ 33; V/VI, n ϭ 42) of the posteromedial barrel subfield of the primary somatosensory cortex of anesthetized rats. Two neighboring principal and adjacent whiskers (PW and AW, respectively) in the same row were deflected rostrally or caudally at varying interstimulus intervals (ISIs). In 37% of the neurons, the response to the combined stimulus was significantly larger than the sum of the responses to stimulation of the individual whiskers. In instances in which response facilitation was observed, selectivity was noted for the combination (75%) of the PW with a particular AW or for a particular direction (60%) of whisker deflection. The direction bias of the responses to multiwhisker stimulation was well correlated with that of the sum of the responses to single whisker stimulation (r ϭ 0.83; p Ͻ 0.001). The pattern and magnitude of the response interaction in the neurons of the superficial layers were closely related to the location of the recorded cell in the barrel columns. Multiwhisker stimulation at short ISIs (Յ3 msec) evoked prominent response facilitation in cells located close to the border between two columns ( p Ͻ 0.05, one-way ANOVA), where two excitatory inputs were expected to arrive at the same time. Our results suggest that the spatiotemporal patterns of multiwhisker stimulation, such as whisker combination, direction of deflection, and timing, are expressed as different magnitudes of response interaction, which depends on the proximity of cells to home and adjacent barrel columns.

During gastrulation in the mouse embryo, dynamic cell movements including epiblast invagination and mesodermal layer expansion lead to the establishment of the three-layered body plan. The precise details of these movements, however, are sometimes elusive, because of the limitations in live imaging. To overcome this problem, we developed techniques to enable observation of living mouse embryos with digital scanned light sheet microscope (DSLM). The achieved deep and high time-resolution images of GFP-expressing nuclei and following 3D tracking analysis revealed the following findings: (i) Interkinetic nuclear migration (INM) occurs in the epiblast at embryonic day (E)6 and 6.5. (ii) INM-like migration occurs in the E5.5 embryo, when the epiblast is a monolayer and not yet pseudostratified. (iii) Primary driving force for INM at E6.5 is not pressure from neighboring nuclei. (iv) Mesodermal cells migrate not as a sheet but as individual cells without coordination.

Most fruit trees in the Rosaceae exhibit self-incompatibility, which is controlled by the pistil S gene, encoding a ribonuclease (S-RNase), and the pollen S gene at the S-locus. The pollen S in Prunus is an F-box protein gene (SLF/SFB) located near the S-RNase, but it has not been identified in Pyrus and Malus. In the Japanese pear, various F-box protein genes (PpSFBB-α–γ) linked to the S-RNase are proposed as the pollen S candidate. Two bacterial artificial chromosome (BAC) contigs around the S-RNase genes of Japanese pear were constructed, and 649 kb around S4-RNase and 378 kb around S2-RNase were sequenced. Six and 10 pollen-specific F-box protein genes (designated as PpSFBB4-u1–u4, 4-d1–d2 and PpSFBB2-u1–u5, 2-d1–d5, respectively) were found, but PpSFBB4-α–γ and PpSFBB2-γ were absent. The PpSFBB4 genes showed 66.2–93.1% amino acid identity with the PpSFBB2 genes, which indicated clustering of related polymorphic F-box protein genes between haplotypes near the S-RNase of the Japanese pear. Phylogenetic analysis classified 36 F-box protein genes of Pyrus and Malus into two major groups (I and II), and also generated gene pairs of PpSFBB genes and PpSFBB/Malus F-box protein genes. Group I consisted of gene pairs with 76.3–94.9% identity, while group II consisted of gene pairs with higher identities (>92%) than group I. This grouping suggests that less polymorphic PpSFBB genes in group II are non-S pollen genes and that the pollen S candidates are included in the group I PpSFBB genes.

lead to a high rate of cardiovascular mortality. In particular, multiple pathways including calcium and phosphorus metabolic abnormalities caused by secondary hyperparathyroidism, inflammation and cardiac overload accelerate the process of valvular calcification, and the consequent P atients with end-stage renal disease (ESRD) on chronic hemodialysis (HD) often have a high prevalence of structural abnormalities of the heart including calcified valvular sclerosis, 1,2 left ventricular (LV) remodeling 3,4 and LV diastolic dysfunction, 5 which may

No abstract

Nanoendoscopy-AFM can visualize the internal cell’s structures in its physiological environment at the nanometer scale.

This protocol describes how to observe gastrulation in living mouse embryos by using light-sheet microscopy and computational tools to analyze the resulting image data at the single-cell level. We describe a series of techniques needed to image the embryos under physiological conditions, including how to hold mouse embryos without agarose embedding, how to transfer embryos without air exposure and how to construct environmental chambers for live imaging by digital scanned light-sheet microscopy (DSLM). Computational tools include manual and semiautomatic tracking programs that are developed for analyzing the large 4D data sets acquired with this system. Note that this protocol does not include details of how to build the light-sheet microscope itself. Time-lapse imaging ends within 12 h, with subsequent tracking analysis requiring 3-6 d. Other than some mouse-handling skills, this protocol requires no advanced skills or knowledge. Light-sheet microscopes are becoming more widely available, and thus the techniques outlined in this paper should be helpful for investigating mouse embryogenesis.