Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator

A stereological method for obtaining estimates of the total number of neurons in five major subdivisions of the rat hippocampus is described. The new method, the optical fractionator, combines two recent developments in stereology: a three-dimensional probe for counting neuronal nuclei, the optical disector, and a systematic uniform sampling scheme, the fractionator. The optical disector results in unbiased estimates of neuron number, i.e., estimates that are free of assumptions about neuron size and shape, are unaffected by lost caps and overprojection, and approach the true number of neurons in an unlimited manner as the number of samples is increased. The fractionator involves sampling a known fraction of a structural component. In the case of neuron number, a zero dimensional quantity, it provides estimates that are unaffected by shrinkage before, during, and after processing of the tissue. Because the fractionator involves systematic sampling, it also results in highly efficient estimates. Typically only 100-200 neurons must be counted in an animal to obtain a precision that is compatible with experimental studies. The methodology is compared with those used in earlier works involving estimates of neuron number in the rat hippocampus and a number of new stereological methods that have particular relevance to the quantitative study of the structure of the nervous system are briefly described in an appendix.