Seasonal plasticity and sexual dimorphism in the avian song control system: stereological measurement of neuron density and number.

Differences in neuron density and number are associated with seasonal plasticity and sexual dimorphism in the avian song control system. In previous studies, neuron density and number in this system have been quantified primarily through nonstereological approaches in thick tissue sections by using the nucleolus as the unit of count. The reported differences between seasons and sexes may be inaccurate due to biases introduced by neuron splitting during sectioning. We used the unbiased optical disector technique on tissue from three previous studies (two investigations of seasonal plasticity and one investigation of sexual dimorphism in avian song nuclei) to assess seasonal and sex differences in neuron density and number. In two song nuclei, HVc and the robust nucleus of the archistriatum (RA), the optical disector yielded intergroup differences in neuron density and number that coincided well with the three previous reports. We also estimated neuron number and density with a random, systematic, nonstereological counting protocol that used the neuronal nucleolus as the unit of count. We compared this method directly to the optical disector. In all cases, the two neuron-counting methods produced similar estimates of neuron number and density; the differences between treatment groups were equally discernible regardless of the counting method used. This study confirms previously reported seasonal and sex differences in the HVc and the RA by use of stereology and indicates that a random, systematic, nonstereological neuron-counting protocol is accurate and is well suited to the study of these phenomena in the avian song control system.