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P21 3h Slice Conditions*

Brain was removed from 21 day-old rat following decapitation and the left hippocampus dissected out. 400 micron slices were cut at 70 degrees transverse to the long axis from the middle third of the hippocampus with a cold tissue chopper and placed into ice-cold physiological saline containing (in mM) 117 NaCl, 5.3 KCl, 26 NaHCO3, 1 NaH2PO4, 2.5 CaCl2, 1.3 MgSO4, and 10 glucose, equilibrated with 95% O2-5% CO2 (pH 7.4). A slice was transferred in this saline, via the blunt end of a glass pipette, directly onto a net over a well with physiological saline, at the interface of humidified 95% O2-5% CO2 at 32 °C in a recording chamber. Total time between death and placement in recording chamber was 7.65 minutes.

Slice was maintained in vitro, undisturbed for 2 hours. After 2 hours, field excitatory postsynaptic potentials were recorded extracellularly to assure slice health prior to fixation. A concentric bipolar stimulating electrode was positioned about 400 microns from an extracellular recording electrode in the middle of stratum radiatum. The slice the stimulus-response curve was sigmoidal and half-maximal responses remained stable for an hour prior to fixation, demonstrating that the slice was healthy.

After a total of 3 hours in vitro, slice was fixed in mixed aldehydes containing 2% paraformaldehyde, 6% glutaraldehyde, 1 mM CaCl2, 2 mM MgCl2, at pH 7.4 for 8 seconds in the microwave oven, which resulted in a final temperature of 29.3 °C. Slice was stored overnight at room temperature in this fixative and then processed for serial electron microscopy using standard procedures.

*All procedures follow NIH Guidelines for the humane care and use of laboratory animals.

Last Updated: 11/2/00