COMPARATIVE-EVALUATION OF SYNAPTOPHYSIN-BASED METHODS FOR QUANTIFICATION OF SYNAPSES

Development, ageing, and a variety of neurological disorders are chara cterized by selective alterations in specific populations of nerve cel ls which are, in turn, associated with changes in the numbers of synap ses in the target fields of these neurons. To begin to delineate the s ignificance of changes in synapses in development, ageing, and disease , it is first essential to quantify the number of synapses in defined regions of the CNS. In the past, investigators have used EM methods to assess synapse numbers or density, but these approaches are costly, l abour intensive, and technically difficult, particularly in autopsy ma terial. To begin to define reliable strategies useful for studies of b oth animals and humans, we used three techniques to measure synaptophy sin immunoreactivity in rat brain. The levels of synaptophysin protein were determined by Western blots of five hippocampal subregions; the intensity of synaptophysin-immunoreactivity in dentate gyrus and strat um oriens was determined by optical densitometry of immunocytochemical ly stained sections; and the total number of synaytophysin-immunoreact ivity presynaptic boutons in dentate gyrus and stratum oriens was asse ssed by unbiased stereology. Each approach has advantages and disadvan tages. Western blotting is the least time-consuming of the three metho ds and allows simultaneous processing of multiple samples. In systemat ically sampled histological sections, both densitometry and stereology allow precise definition of the region of interest, and the stereolog ical optical disector method allows quantitation of the numbers of syn aptophysin-immunoreactive boutons. Stereology was the only method that clearly demonstrated greater synaptophysin-immunoreactivity in the de ntate gyrus as compared to stratum oriens. The use of systematic sampl ing and the disector technique offer a high degree of anatomical resol ution (lacking in Western blot methods) and has quantitative advantage over the greyscale-based density approach. Thus, at present, stereolo gy is the most useful method for estimating synaptic numbers in define d regions of the brain.