INSITU HYBRIDIZATION HISTOCHEMISTRY QUANTIFICATION - AUTOMATIC COUNT ON SINGLE-CELL IN DIGITAL IMAGE

It is extremely useful in investigations of the central nervous system (CNS) to measure mRNA expression in cells by in situ hybridization. H owever, this approach is limited by the difficulties of a reliable qua ntitative evaluation. In the present paper we describe a method for qu antifying radioactive hybrids on individual cells by a silver grain co unt in digital images. Quantification is based on the real size and gr ey level of a single grain obtained by computerized microscope image a nalysis (IBAS 2, Kontron-Zeiss, PC 286). The program provides for auto matic identification of cell area, the portion occupied by grains and their grey level. The number of grains per cell results from a mathema tical function integrating these parameters with a 'density factor'. T his factor is introduced to better estimate the number of grains when there is overlapping. The method was tested by measuring the expressio n Of preproNPY (pp-NPY) mRNA in rat dentate gyrus and preproSomatostat in (pp-SOM) mRNA in frontal cerebral cortex of control and colchicine- treated rats. Colchicine did not modify the number of pp-SOM mRNA-posi tive cells but reduced the expression per cell. These results confirm the advantages of our method to quantify a wide range of silver grains (10-5000) and improves the sensitivity of in situ hybridization. With this support the in situ hybridization technique could be considered a real quantitative method for measuring small alterations in neuronal function.