Computational protocol: Postsynaptic adhesion GPCR latrophilin-2 mediates target recognition in entorhinal-hippocampal synapse assembly

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Protocol publication

[…] For neuron cell culture experiments, dendritic morphology was visualized by sparse calcium phosphate transfection of a GFP-expressing construct (L316). Neurons were selected for imaging and analysis based on pyramidal neuron morphology exhibited by prominent apical dendrites as well as the presence of spine structures protruding off dendrites. Images were acquired using a 60× objective with 4× digital zoom on an A1 Eclipse Ti confocal microscope with constant image settings operated by NIS-Elements Advanced Research v4.5 acquisition software (Nikon). Z stack images were converted to maximal projection images and analyzed using MetaMorph Software (Molecular Devices) with synaptic puncta quantified for puncta density per 10 µm of dendrite, size, and intensity. [...] 2–3-mo-old mice were put through a modified version of a Barnes maze protocol described previously (). The maze consists of a brightly lit circular open platform (92 cm diameter) with 20 equally spaced holes (hole diameter, 5 cm) along the perimeter. Underneath the designated target hole, an escape box (7 cm deep, 7 cm width, and 10 cm length) was placed. Underneath the remaining holes, false escape boxes lacking depth were placed, made of same color/texture material as the escape box. Extra-maze cues were placed on the surrounding walls to serve as reference cues to learn the position of the target escape hole. To begin the maze, mice were placed in the center of the maze in a holding chamber (15 cm × 15 cm) for 30 s. The chamber was then lifted, and the mice were free to explore the maze with 19 of the 20 holes closed and were assayed for their ability to spatially navigate the maze to find the target escape hole. The target escape box was positioned underneath the maze as a small dark recessed chamber, which the mice naturally sought out, taking advantage of their desire to escape brightly lit and exposed environments. During the initial four-consecutive-day training period, the mice learned the spatial location of the target hole, with four trials conducted per day (∼2-h intertrial interval). Parameters measured included the time to target hole (latency) as well as time spent within the target hole (affinity). Memory of the target hole was measured as a single trial, conducted at 1 d and 14 d after the initial training period. Data acquisition and analysis was performed using BIOSERVE Viewer v6.1 video tracking software (VMware). […]

Pipeline specifications

Software tools MetaMorph, VMware
Applications Miscellaneous, Laser scanning microscopy
Organisms Mus musculus