The use of the optical disector to estimate the total number of neurons in the developing chick lateral motor column: Effects of purified growth factors

Abstract

Competition between neurons for limited amounts of trophic factors is believed to be the basis for large-scale neuronal death during the normal development of the vertebrate nervous system. In this study, an unbiased stereological counting method, an optical disector/Cavalieri combination, was used to estimate the total number of motor neurons in the lateral motor column of the developing chick and to assess the effects of four growth factors on neuronal numbers. The total number of neurons in lateral motor columns at embryonic day 6 (E6), E8, E10 and E12 were 18,747 +/- 1,369 (mean +/- SD), 15,037 +/- 1,816, 10,245 +/- 940, and 8,802 +/- 797, respectively. Daily exposure from E6 to E9 to three of the growth factors (basic fibroblast growth factor, bFGF; leukemia inhibitory factor, LIF; nerve growth factor, NGF) had no effect on total neuron number at E10. However, exposure to ciliary neurotrophic factor (CNTF) from E6 to E9 significantly increased (P less than 0.05) the number of neurons in the lateral motor column (13,610 +/- 725, compared with 10,058 +/- 204 in normal saline controls). These results are in agreement with previous reports of large scale neuronal death in the developing chick lumbar lateral motor column between E6 and E12 and confirm that exposure to growth factors such as CNTF can mitigate the course of normal ontogenetic cell death. The optical disector/Cavalieri combination is an efficient method for counting neurons: on average, following sectioning and staining, less than 30 min was required to estimate the total number of motor neurons in a lateral motor column with a coefficient of error of approximately 10%.