The Macro-molecule Communication Or Transport Between Hippocampal Neurons

Objective To investigate the ultrastructure of the connection between two primarily cultured hippocampal neurons with atomic force microscopy (AFM), and whether there is macro-molecule communication or material transport between two primarily cultured hippocampal neurons. Methods The C57 milk mice within 12 h after birth were anesthetized, sterilized and decapitated. The skull was cut and the brain was taken out. The hippocampus was dissected under an anatomy microscopy and cut into pieces. Primary hippocampal neurons were cultured. The Well-growing hippocampal neurons chosen under light microscopy was fixed for 30min by 2.5% glutaraldehyde of normal saline solution. The crystals of NaCl and other materials were removed by flowing triply distilled water. The culture dish with the interesting structures was rapidly cut into appropriate sheets and immobilized onto the base of AFM, which was then put onto the top of the AFM scanner. AFM imaging was carried out by using contact mode in air at room temperature (22-24℃). Each of the RFP C57milk mice and EGFP C57milk mice within 48h after birth was anesthetized, sterilized and decapitated. The skull was cut and the brain was taken out. The hippocampus was dissected under an anatomy microscopy and cut into pieces. Primary hippocampal neurons were co-cultured. Well-growing hippocampal neurons were chosen to be studied under the fluorescence microscope. Results Hippocampal neurons displayed smooth membrane surface with even and regular undulation, the neurites interlaced network structures and the neurons formed synapse-like structures with each other. The soma of the neurons were divided into two (upper, lower) or three (upper, middle, lower) parts by one or two horizontal grooves. All of the neurites were from the upper parts and none from the middle and the lower parts. There are continuous membranous junctions (CMJ) between two closely adjacent neurons and non-synaptic neurite connections (NSNC) between two remote neurons. There are both green fluorescent protein and red fluorescent protein in one of the two closely adjacent neurons connected with the continuous membranous junctions (CMJ). Conclusions The results once again demonstrated that AFM is a useful tool to study the subcellular morphology of cultured neurons. We confirmed those unexplainable connection structures completely different from synapse:(1) CMJ between two closely adjacent neurons; (2) NSNC between two remote neurons. And we also deduced there may be certain macro-molecule communication or material transport in the CMJ.