Proximity of excitatory and inhibitory axon terminals adjacent to pyramidal cell bodies provides a putative basis for nonsynaptic interactions. Video sequence made up of 14 consecutive confocal microscope images (z-step: 0.14 m) from a pyramidal cell in layer II of the mouse visual cortex. Vesicular glutamate transporter 1 (VGluT1; marker for glutamatergic terminals; green) and GABA transporter 1 (GAT1; marker for GABAergic terminals; red) are present in punctate structures in the neuropil, and around the cell body and dendritic trunks (unlabeled areas). VGluT1- and GAT1-positive profiles are frequently apposed to one another and to the cell soma. Although there is no colocalization of these markers, a yellow overlapping area is visible in some cases, when both kinds of terminals are closely apposed one on top of the other. This z-series is from the same pyramidal cell as Fig 1G in Proc Natl Acad Sci. (2009) 106: 9878-9883.
Five C57 male mice 26 days of age were administered a lethal i.p. injection of sodium pentobarbital (40 mg/kg) and then were intracardially perfused with saline solution followed by 4% paraformaldehyde in 0.12M phosphate buffer, pH 7.4 (PB). The heads were left overnight in the same fixative at 4C, and the brains were removed the next morning and postfixed in fresh fixative for 1 additional day. Thereafter, the brains were washed in several changes of 0.12 M PB and free-floating vibratome sections (100-μm thick) were used for these experiments. Primary antibodies used were: guinea-pig anti-VGluT1 (Millipore Corp) and rabbit anti-GAT1 (Millipore Corp). Primary antibodies were diluted in PB containing 0.25% Triton X-100. Secondary antibodies were: anti-guinea-pig Alexa 488 and anti-rabbit Alexa 594. The sections were photographed on a Leica DMI 600B laser scanning confocal microscope, and the visualization and deconvolution of the images was performed with Imaris (Bitplane AG) and Autodeblur (Media Cybernetics Inc.) softwares.
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