Development of Quantitative Methods for the Evaluation of the Entity of Coexistence of Neuroactive Substances in Nerve Terminal Populations in Discrete Areas of the Central Nervous System: Evidence for Hormonal Regulation of Cotransmission

Abstract

We have recently developed several methods for the quantitative evaluation of coexistence of neuroactive substances in nerve cell bodies, namely the overlap method and the occlusion method (Agnati et al. 1982, Agnati et al. 1983, Fuxe et al. 1982, 1983, Agnati et al. 1984). In the overlap method the image analyzer can record the position and the perimeter of each nerve cell body visualized by means of the antiserum anti A in a Cartesian plan. Using an adjacent thin section or the same section following mild elution of anti A immunoreactivity the staining against antibody B is performed. The image analyzer then again records the position and the perimeter of the nerve cell bodies showing B immunoreactivity in the same Cartesion plan as used for the demonstration of anti A immunoreactivity. The positions in the Cartesian plan of the neurons are then compared by the computer and coexistence is considered to take place when an anti-A immunoreactive area and an anti-B immunoreactive area overlap by at least 30%. Thus, a 30% overlap is considered to be a threshold value below which coexistence is not considered to exist. When using thin adjacent sections a correction factor must be calculated since some A and B immunoreactive nerve cell bodies disappear as they move from one section to the other.