Bitan:Dendritic spine morphology

Study of dendritic spine morphology

Rat primary hippocampal neurons were used to study the dendritic spine morphology

Reagents and accessories:

DiL stain, streptomycin/penicillin (Invitorgen), paraformaldehyde, Poly D-lysine, cytosine β-D arabinoside (Sigma), neurobasal media, B27 factor (GIBCO), cover slip, capillary glass tube (Fisher Scientific).

Protocol:

Hippocampi were dissected out from E18 pups and cultured for three weeks in neurobasal medium supplemented with B27.

After three weeks, the cells were treated with peptides (3μM) for 3 days.

After treatment the cells were fixed with 4% paraformaldehyde and stained each individual neuron with DiI by microinjection (Eppendorf Cell Technology, Model: 5070) method.

The DiI labeled neurons were imaged at high magnification (100X oil-immersion objective) using the Leica confocal laser-scanning microscope.

Images were magnified further using a 3X zoom so that the morphology of individual spines could be determined and subsequently quantified.

The Z stack images were collected at 0.3 μm intervals to cover the full depth of the dendritic arbors (20-30μm) and then compressed into a single JPEG image. The numbers of dendrtic spines were counted in 100 μm lengths of dendritic branches using Image J software