Analysis of cell death in the trochlear nucleus of the chick embryo: Calibration of the optical disector counting method reveals systematic bias

Detection of changes in numbers of neurons is essential for an understandin g of neuronal development, function, and death. Optical disector counting i s claimed to be the most efficient technique to estimate accurate numbers o f neurons in microscopic sections. We calibrated the optical disector by co mparison with three-dimensional reconstructions from serial sections and de termined how accurate this technique is relative to conventional profile co unting methods. The calibration was performed on the trochlear nucleus in d eveloping chicks. Optical disector estimates, when obtained as generally re commended, were about 25% lower than the actual number of neurons. This und erestimate was caused by a nonuniform (bimodal) distribution of neuronal nu clei in paraffin and plastic (glycolmethacrylate) sections, but not in cryo sections. The density of neurons in the core of the paraffin and plastic se ctions was substantially lower than in the upper and lower margins of these sections. Accurate estimates of neuronal numbers were obtained with a modi fied optical disector method that sampled the entire extent of tissue secti ons. Previous estimates of numbers of trochlear neurons in the developing c hick have been controversial. The modified (calibrated) optical disector me thod revealed that the number of trochlear neurons decreased from about 1,6 00 at day 8.5 of incubation (embryonic day, [E]8.5) to about 900 at the tim e of hatching. Numbers of pyknotic nuclei peaked at E6 and at E9, revealing an additional early, but postproliferative, period of cell death. Taken to gether, these data emphasize the need for calibration of stereological coun ting techniques and the need to examine sampling strategies for potential b ias. (C) lass Wiley-Liss, Inc.