Yukako Yamane

Complex objects are represented in macaque inferotemporal cortex by the combination of feature columns

Intrinsic signal imaging from inferotemporal (IT) cortex, a visual area essential for object perception and recognition, revealed that visually presented objects activated patches in a distributed manner. When visual features of these objects were partially removed, the simplified stimuli activated only a subset of the patches elicited by the originals. This result, in conjunction with extracellular recording, suggests that an object is represented by a combination of cortical columns, each of which represents a visual feature (feature column). Simplification of an object occasionally caused the appearance of columns that were not active when viewing the more complex form. Thus, not all the columns related to a particular feature were necessarily activated by the original objects. Taken together, these results suggest that objects may be represented not only by simply combining feature columns but also by using a variety of combinations of active and inactive columns for individual features.

Three-dimensional characterization of interior structures of exocytotic apertures of nerve cells using atomic force microscopy

We examined the interior structure of exocytotic apertures in synaptic vesicles of neuroblastoma x glioma hybrid cells using atomic force microscopy. The atomic force microscopy detected apertures of 50-100nm in diameter at various depths within the varicosities of these cells. We were also able to image a regular radial pattern on the wall and lump-like structures at the bottom of these apertures. In contrast, scanning electron microscopy could only detect the apertures but not the fine details of their interior. The cells examined here exhibited the same electrophysiological properties and expression of synaptophysin and syntaxin 1 as presynaptic terminals, as studied by various electrophysiological and imaging techniques. Our results indicate that atomic force microscopy allows three-dimensional viewing of the fine structures located inside exocytotic apertures in nerve cells.

Acquisition of neuronal proteins during differentiation of NG108-15 cells.

The differentiated type of neuroblastomaxglioma hybrid cell line, NG108-15, has widely been used in in vitro studies instead of primary-cultured neurons. Here we examined whether NG108-15 cells can be used as a model for studying the neuronal differentiation process. We compared the expression of neuronal proteins (neurofilament 200 (NF200), phosphorylated-NF200 (p-NF200), microtubule associated protein 2, synaptophysin, syntaxin 1, choline acetyltransferase, and acetylcholinesterase (AChE)) and a glial protein (vimentin) between undifferentiated and differentiated NG108-15 cells by immunocytochemistry and immunoblot analysis. The expression of all neuronal proteins, with the exception of NF200 and p-NF200, was positive in differentiated cells, but almost negative in undifferentiated cells. On the other hand, cytoskeletal intermediate filaments (NF200 and p-NF200) for neurons and that (vimentin) for glia were present in both undifferentiated and differentiated cells. Furthermore, a high expression of AChE mRNA was confirmed in differentiated cells by reverse transcription-PCR analysis. Our results showed that even though the expression of cytoskeletal filaments does not change during differentiation of NG108-15 cells, these cells during differentiation can serve as an appropriate tool for investigating and understanding the mechanisms involved in neuronal development and differentiation.

GAP junctional channel inhibition alters actin organization and calcium propagation in rat cultured astrocytes.

Astrocytes are connected by gap junctions, which provide intercellular pathways that allow a direct exchange of ions and small metabolites including second messengers and the propagation of electric currents. The roles of gap junctional communication on whole-cell morphology, cytoskeletal organization, and intercellular communication in astrocytes are not yet clear even in vitro, though there are many studies that have examined the active relation between gap junctions and actin filaments in astrocytes. Here we examined the effects of gap junction inhibitors, which do not interrupt the formation but rather the function of gap junctions, on whole-cell morphology, cytoskeletal organization, and intercellular communication in rat cultured astrocytes. Functional blockade of gap junctions during the formation of an astrocytic monolayer resulted in discordance of actin stress fibers between neighboring cells, even though whole-cell morphology of these cells did not change by such treatment. Mechanical stimulation-induced calcium wave propagation was significantly reduced in these actin-discordance cells even after thorough wash out. Differentiation of astrocytes in the presence of gap junction inhibitors was associated with morphological disarrangement among neighboring cells due to disordered alignment of actin stress fibers between cells.Our results indicate that gap junctional communication enables cell-to-cell coordination of actin stress fibers in astrocytes, thus enhancing intercellular communication through calcium spread.

Comparative Atomic Force and Scanning Electron Microscopy for Fine Structural Images of Nerve Cells

Although we can routinely obtain fine structural images of cells by atomic force microscopy (AFM), the adequacy and reliability of morphological information acquired from these AFM images remain to be examined. In this report, we compared images of the fine structures of nerve cells as observed by both AFM and scanning electron microscopy (SEM). Although AFM revealed the structure of the top views of cells in greater detail than SEM, their side structures were better observed by SEM. The linear structures in the neural processes detected only by AFM were confirmed, by immunofluorescence staining, to be reflections of the cytoskeletal structures located beneath the cell membrane. These differences between the AFM and the SEM images reflected the characteristics of the detection systems and methods used for sample preparation. Therefore, these results revealed that more detailed information on cell morphology can be obtained by using both AFM and SEM to advantage.

Mechanical Properties of Membrane Surface of Cultured Astrocyte Revealed by Atomic Force Microscopy

In order to examine the mechanical properties of the membrane surface of astrocytes, we observed living astrocytes by atomic force microscopy (AFM) both in contact mode and force-mapping mode. Ridge-like structures reflecting actin filaments were observed in the topographic images in contact mode, but not in force-mapping mode, using a zero-loading force. When we measured the elasticity of astrocytes, we observed that the cell membrane above the nucleus was soft and the cell membrane above the cytosol was stiff. In particular, the parts reflecting actin filaments were very stiff. This effect of actin filaments on the elasticity of astrocytes was confirmed by the loss of actin filaments after application of actin-polymerization inhibitor.

Fine Surface Images That Reflect Cytoskeletal Structures in Cultured Glial Cells by Atomic Force Microscopy

The morphology of cultured glial cells was examined using a combination of atomic force microscopy (AFM) and immunofluorescence staining for cytoskeletons. The meshwork of type-1 astrocytes consisted of thick longitudinal and thin lateral lines on the cell surfaces observed by AFM; the former lines were confirmed to be reflections of actin filaments. The astrocytic processes of type-2 astrocytes were observed to be rugged on AFM. These structures were mainly affected by microtubules. Immunofluorescence imaging of microglia revealed that actin filaments and microtubules were arranged radially and wavily along the cell edge, respectively. AFM could detect these radial and wavy structures clearly. These results show that AFM can provide information on the cytoskeletons of glial cells, indicating that AFM is a useful tool for the morphological characterization of cells.

Atomic Force Microscopic Observation of Three-Dimensional Morphological Changes of Neurons When Stimulated by a Neurotransmitter

As the first step in the study of morphological changes in neurons associated with their functional changes, we applied atomic force microscopy (AFM) for the observation of fine three-dimensional morphological changes in rat cerebellar granule cells stimulated by an agonist of glutamate receptors, N-methyl-D-aspartate (NMDA). The AFM revealed that NMDA changed the cross-sections of cell bodies from a trapezoid-like form to a triangle-like form within a minute. The fine hill-like structures on the top surfaces of the cell bodies became wider during the same period. These results were suggested to be induced by the depolymerization of filamentous actin triggered by the entry of Ca2+ via cation channels complexed with the activated NMDA receptors.

Surface Structures of Cultured Type 2 Astrocytes Revealed by Atomic Force Microscopy

Abstract The incomplete morphological characterization of type 2 astrocytes is in part responsible for the slow progress of studies on these cells. To examine and characterize type 2 astrocytes morphologically, three-dimensional fine structures of the surfaces of type 2 astrocytes cultured from rat cerebella were studied by a combination of atomic force microscopic and immunocytochemical techniques. Atomic force microscopy (AFM) revealed irregular ridge-like structures that form a meshwork distributed throughout the cell body surfaces and the thick processes. These ridges were found to be of two heights (31 nm and 82 nm). This finding indicates two possible configurations responsible for shaping the meshwork: (1) two structures of different thickness are beneath the cell membrane; and (2) two structures are located at two different depths from the cell membrane. On the other hand, immunocytochemical studies for tubulin and glial fibrillary acidic protein (GFAP) revealed that these cytoskeletal filaments are similarly distributed within the resolution power of a light microscope. However, no detectable structures were obtained by actin staining. The immunocytochemical findings suggest that the AFM-revealed ridges forming the irregular meshwork on the cell surfaces may reflect very fine bundles of tubulin and/or GFAP. Therefore, AFM study, with the help of immunocytochemical study, is a powerful tool for characterizing cell morphology. The results of the present study reveal the first morphological characterization of type 2 astrocytes.

How Images of Objects Are Represented in Macaque Inferotemporal Cortex

Visual object recognition is a simple and easy task in our daily life. However, the mechanisms for recognizing objects are not at all simple nor easy. To understand neural mechanisms of object recognition, we have investigated rep- resentation of object images in macaque inferior temporal cortex that is the area essential for object recognition. Optical intrinsic signal imaging has revealed that object images are represented by the combinatorial code at the columnar level, where each column represents a visual feature of object images. The visual features represented by columns include local features as well as global features repre- senting spatial arrangements of local features. Here, columns are supposed to be functional units for object representation. However, difference in object selectivity among nearby cells does not support the concept of columns as the functional units. Quantitative analysis of object responses of single cells and population activity revealed that each cell in a columnar region is characterized by cell specific prop- erty and property common across the cells in the columnar region, suggesting two different levels (single cell and columnar level) of object representation. Possible role of these two levels of object representation will be discussed.