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M. Ellisman, M. Martone, N. Yamada (Univ. Calif. SD, NCMIR)

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$B!!(BMany neuronal networks in the mammalian central nervous system provide direct cell-to-cell communication, conduction of ionic currents and passage of small organic signaling molecules, through electrical synapses. The vast majority of these synapses are gap junctions, specialized intercellular contactswithaggregates of transmembrane channels composed of a family of protein subunits termed connexins. Study of electrical synapses needs comprehensive evidence showing functional significance by simultaneous dual patch-clamp recordings between neighboring cells of certain neuronal type and structural identification of gap junctions between the examined cells. Such studies are considered to be less copious and in fact might be relatively rare.

$B!!(BIn retinal ganglion cells, the occurrence of electrical synapses between neighboring cells has been proposed by electrophysiological recordings of distributed spikes. For a specific mammalian cell type, a-type ganglion cells ($B&A(B-GCs), synchronous spike activity has been described in the cell population. In the present study, electrical synapses between $B&A(B-GCs were detected usingcombined techniques of dual recordings, high-voltage electron microscopy and connexin immunocytochemistry in rat retina. After intracellular injection of Neurobiotin into $B&A(B-GCs of inner (ON-center) and outer (OFF-center) ramifying types, measurement of tracer coupling resulted in preferentially homologous occurrence among cells of the same morphological type. In high-voltage electron microscopic analysis of 5-$B&L(Bm-thick sections, direct dendrodendritic gap junctions (average size 0.86 $B&L(Bm long) were present in contact sites between tracer-coupled $B&A(B-GCs. In simultaneous dual recordings from pairs of neighboring $B&A(B-GCs, bidirectional electrical synapses and precise temporal synchronization of spike activity were detected in pairs with cells of the same morphological type. To address whether physiologically identified electrical synapses constitute gap junctional connectivity between cell pairs, connexin36 immunoreactivity was undertaken in Lucifer yellow-labeled cell pairs after patch-clamp recordings. Confocal laser-scanning imaging demonstrated that connexin36 is located at dendritic crossings between electrically coupled cells. These results give conclusive evidence for electrical synapses via dendrodendritic gap junctions in $B&A(B-GCs of the same physiological type.

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$B!!(BHidaka, S,Akahori, Y, & Kurosawa, Y (2004)Dendrodendritic electrical synapses between mammalian retinal ganglion  cells. Journal of Neuroscience, Vol 24 (No 47) : pp 9718 - 9732.

Figure 1.	A, Fluorescence photomicrograph of two $B&A(B-GCs of the same type in a whole-mount retinal preparation. These cells were filled with Lucifer Yellow under simultaneous dual patch-clamp recordings. The pair of $B&A(B-GCs showed bidirectional electrical synapses in current clamp condition.
	B, Electron micrograph showing the direct junctional contact between Neurobiotin-coupled neighboring $B&A(B-GCs of the same type. The occurrence of the connection was revealed by high-voltage electron microscopy at 1,000 kV in a tangential 5-$B&L(Bm-thick section. The dendrodendritic gap junction (arrows) occurs between the tips of peripheral dendrites from coupled $B&A(B-GCs.

 

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11. 3-D Reconstruction of the Crystalline Bodies and the Rhabdomere Formation during Development

InSun Kim(Keimyung University)
Sung-Sik Han (Korea University)

$B!!(BImage processing by UHVEM and electron tomography has offered major contributions to research areas of both cellular and subcellular levels. Furthermore, such advancements have enabled improved analysis of 3-dimensional cellular structures.

$B!!(BThe late pupal stage of Drosophila melanogasteroccurs immediately before the completion of retinal development, during which the rhabdomere rapidly forms. In this period, the photoreceptor cells were fixed and dehydrated using a high-pressure freezer (HPF) /freeze-substitution (FS) technique, which is the most effective in preserving the cell structures, and observed using high-voltage electron microscopy (HVEM) at 1000 KV.

$B!!(BThe results suggest that there are at least three types of vesicles related to rhabdomere formation in photoreceptor cells. In addition, it was found that these vesicles initiate the formation of the rhabdomeres during the pupal stage. The rhabdomere is classified structurally into three types of formation patterns using tilt images (fig 1), and stereo-tiling image (fig 2). Initially, hexagonal arrays of rhabdomere existed in different angles. In addition, small pieces of rhabdomeres can be observed in the cytoplasm of the photoreceptor cells, which are visible during the process of rhabdomere formation. In addition, multiple layers of rhabdomere strings were observed. It was also observed that rhabdomeres were mainly formed through vesicles, and that parts of the rhabdomere formed first and then gathered and formed rhabdomeres in the late pupal stage.

Fig 1.	In order to create a tilting movie, a total of 60 images were taken by tilting a thick-sectioned ribbon by 2$B!k(B. Images presented in this figure were 6$B!k(Bapart.	Fig 2.	Stereo-pair is presented. When observed with  a stereo-viewer, rhabdomere formation does   not occur at only one place in the cytoplasm of the photoreceptor cells. Membrane particles, which are believed to be membrane proteins, are clearly identified in the membrane of membrane-bounded vesicles, and their vesicles are fused into rhabdomeres being formed.

 

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