(a-d) Pregnant mice were injected with BrdU on E12.5 (a, a1, a2, and b)
or E16.5 (c, c1, c2, and d). Whole mouse brains were harvested on P20. (a-a2 and
c-c2) Coronal brain sections through the DCN were double stained for neuN
(green) and BrdU (magenta) and imaged by a confocal microscope. Large neurons
are indicated by white arrows and small neurons by white arrowheads. Most
neurons born on E12.5 (the peak of the first neurogenesis) have large sizes
(average soma diameter = 16.03 ± 0.59 μm, n = 30 BrdU-labeled
neurons from three animals) while those neurons born on E16.5 (the peak of the
second neurogenesis) have small sizes (average soma diameter = 7.55 ±
0.20 μm, n = 52 BrdU-labeled neurons from three animals). (b) Double
immunostaining of NR2B (green) and BrdU (magenta) within the DCN indicates that
the large neurons born on E12.5 are glutamatergic excitatory neurons. (d) Double
immunostaining of GABA-B receptor 1 (green) and BrdU (magenta) within the DCN
demonstrates that the small neurons born on E16.5 are GABAergic local circuit
neurons. The axis in (a) indicates the orientation of all the sections in the
figure. D, dorsal; L, lateral. Scale bar in (d) is 30 μm.