Quantitative analysis of neuronal mosaic formation in the mouse neocortex and hippocampus

Abstract

The mosaic pattern of neurogenesis, reflected in the arrangement of concentrations of neurons intensely labeled with 3H-thymidine, was described as a result of the writers' investigations with injection of 3H-thymidine into mice at different times of embryogenesis, followed by analysis of the arrangement of the labeled cells in the neocortex and hippocampus of day-old mice [4, 5, 13]. It was concluded that the mosaic pattern of neurogenesis is determined by the nonsynchronized course of differentiation of cell groups in the ventricular zone of the embryonic brain, i.e., evidence was obtained of its discrete organization in the form of loci of neurogenesis. The aim of the present investigation was to obtain mathematical confirmation of the nonrandomness of mosaic formation of neuronal groups in the cerebral cortex, to characterize this process quantitatively, and to compare its scale with the formation of the modular organization of the cortex. EXPERIMENTAL METHOD Pregnant female CBA mice were given a single intraperitoneal injection of 3H-thymidine (i0 ~Ci/g) on the 13th-19th day of pregnancy (EI3-EI9). At the age of i day, two or three mice from each mother were killed by decapitation. The cerebral hemispheres of mice receiving 3H-thymidine from El5 to El9 were fixed in Karnovsky's fixative and embedded in Durcupan. When the isotope was injected during the period E13-19 the cerebral hemispheres were fixed in Carnoy's mixture and embedded in paraffin wax. One hemisphere from each animal was cut into frontal sections, the other into sagittal. Preparations with glued semithin (i u) or paraffin (6 B) sections were covered with type M emulsion and, after standard autoradiographic processing, were stained with 1% toluidine blue solution in 2.5% sodium carbonate solution or with 0.1% cresyl violet. The arrangement of the centers of the nuclei of the intensely labeled neurons in area 6 of the frontal region of the neocortex and in area CAI of the dorsal hippocampus was mapped on an NU-2E microscope with projection screen. Mapping in the neocortex was carried out on frontal semithin brain sections from mice receiving 3H-thymidine from El5 through EfT, whereas mapping in the hippocampus was carried out on paraffin and semithin frontal brain sections from animals receiving the isotope from El3 through El7. Cells were recorded as intensely labeled if the number of grains of silver above the nucleus varied from maximal to half that number of grains per section. Maps of areas 6 and CAI from two animals at each time of the experiment studied were analyzed mathematically to discover nonrandom groups of intensely labeled neurons. For this purpose an approach was used which enables the regularity of neurogenesis to be assessed in brain structures organized on the principle of a rec