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. 1998 Dec 1;18(23):9910-23.
doi: 10.1523/JNEUROSCI.18-23-09910.1998.

Neurogenesis and Commitment of Corticospinal Neurons in Reeler

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Free PMC article

Neurogenesis and Commitment of Corticospinal Neurons in Reeler

F Polleux et al. J Neurosci. .
Free PMC article

Abstract

In the homozygous (but not the heterozygous) reeler mutant, disruption of neuron migration leads to a major perturbation of the cortical environment that in turn could modify (1) the specification of neuronal fate and (2) the proliferation dynamics of cortical precursors. To investigate these issues, tritiated thymidine injections during cortical neurogenesis were coupled with postnatal injections of a retrograde tracer in the spinal cord to accurately measure the neurogenesis of corticospinal neurons in the heterozygous and homozygous mutant. The homozygous reeler shows (1) strict conservation of area-specific timetables of corticospinal neuron generation; (2) neurons with the appropriate birthdates show an enhanced probability of projecting to the spinal cord; (3) during early stages of corticogenesis, there is a reduced rate of neuron production followed at later stages by an increased rate of neuron production; and (4) these changes in the rate of neuron production were shown to be at least partially attributable to changes in the proportions of differentiative divisions. Taken together, our results show that in the developing cortex, the neurogenesis and specification of a given neuronal phenotype are partially controlled by the postmigratory compartment. On the other hand, neither areal identity nor the chronology of production of layer-specific neuronal phenotype seems to depend on the integrity of the cellular environment.

Figures

Fig. 1.
Fig. 1.
Microphotographs showing NADPH diaphorase labeling in the cortex of normal (A) andreeler mice (B) on a coronal section taken at the rostrocaudal level indicated in theinset (C). In normal mouse, NADPH diaphorase-rich neuropil delineates the primary somatosensory area 3, where individual barrels can be seen in the barrel field. In thereeler mouse neocortex, patches of high NADPH–diaphorase activity spanning the middle two-thirds of cortical thickness delineate the barrel field. The medial limit to area 3 is equally sharp in reeler and normal. Scale bar (shown inA for A, B): 500 μm.
Fig. 2.
Fig. 2.
Cytoarchitectonic analysis of the normal andreeler mouse neocortex. Cell bodies were stained using cresyl violet, and all identifiable neuronal profiles encountered in a radial strip of primary somatosensory cortex were drawn.A, Normal mouse; B, reelermouse. All neuronal profiles are shown in the far left panel. The following six panels show the radial distribution of neurons showing different categories of diameters. C, Histograms representing the mean number of neurons for each category encountered under 1 mm2 of cortical surface in parietal area 3; D, total mean number of neurons encountered under 1 mm2 of cortical surface. Data were collected from seven normal and seven reeler mice. In all cases, error bars indicate 1 SD. Statistical analysis: *p < 0.05; ns, not statistically significant according to a Mann–Whitney U test.gn, Region where granular neurons are encountered in thereeler cortex. Scale bar (shown in A forA, B): 45 μm.
Fig. 3.
Fig. 3.
Low-power montage of microphotographs showing the distribution of corticospinal neurons in adult normal (A) and reeler(B) in cortical areas 3, 4, and 6. Area 4 is located between area 3 (to the left) and area 6 (to theright). Microphotographs from rostrocaudal level shown in Figure 1C. The plots shown in C–Fillustrate more closely the differences in radial distribution of corticospinal neurons in area 6 in normal (C) andreeler (D) and in area 3 in normal (E) and reeler(F). Histograms of the radial distribution of corticospinal neurons taken from four to five sections in two to three adult cortices are shown for reeler in area 6 (G) and area 3 (H). To construct these histograms the cortex has been divided into 10; bin 1 is superficial, bin 10 is deep. Scale bar (shown in Bfor A, B): 150 μm.
Fig. 4.
Fig. 4.
Low-power plots of FG neurons labeled by successive injections of tritiated thymidine spanning the period of cortical neurogenesis in area 6 (A) and area 3 (B) in normal and reeler mouse. The level of sections examined is indicated in the insetin Figure 1. Each 500-μm-large stripe represents the cumulation of two sections taken from two different animals from the same litter. Cortical layers are indicated with roman numerals.C, D, Box plot representation of the radial distribution of area 3 neurons born on E15.5, E16, and E17.5 in normal (C) and reeler cortex (D). Each box plot is formed from a gray box indicating the median and the 25th and 75th percentiles. Error bars indicate the 10th and 90th percentiles. This analysis illustrates the major features of the radial distribution of neurons born on different dates.
Fig. 5.
Fig. 5.
Timing and tempo of generation of corticospinal neurons. A, Developmental changes of the radial position of FG neurons born on E13.5, E15.5, and E16 with respect to corticospinal neurons. Small ×: retrogradely labeled CSNs; red dots, FG neurons; red stars, DLNs (i.e., corticospinal–FG neurons). B, C, Cumulative representation of the generation rate (DLN/CSN) where the cumulated number of DLN produced during the period of corticospinal neurons production divided by the total number of corticospinal neurons is taken as 100%. This measure provides a temporal description of corticospinal neuron production in normal (B) and reeler(C). D, E, Comparison of percentages of DLN/CSN in reeler and normal in area 6 (D) and area 3 (E). Statistical analysis: *p < 0.05, **p < 0.01, according to a χ2analysis. PS, Pial surface; WM, white matter/gray matter limit. Scale bar, 500 μm.
Fig. 6.
Fig. 6.
Developmental changes in the fate index (i.e., percentages of double-labeled neurons with respect to FG neurons) observed in adult (A) and neonates at P6 (B). In A and B, the DLN/FG neuron ratio has been calculated for neurons encountered in a 1-mm-large stripe of cortex comprising areas 6, 4, and 3.A, Comparing adjacent percentages, the χ2analysis reveals a significant and progressive increase in the DLN/FG neuron ratio between E13.5 and E15.5, which suggests that in the normal animal, the probability for a cortical neuron to project to the spinal cord depends largely on its date of birth. Above each bar, then value indicates the total number of FG neurons examined to calculate the ratio. B, The χ2analysis shows that a higher proportion of neurons born on E14.5 express this corticospinal neuron phenotype in thereeler than in the normal mouse. For each bar, thesmall symbols indicate the mean value of one animal from either one litter (E13.5, E16, or E17.5) or from two different litters (E14.5) (▾ and •). In B, the total number (n) of FG neurons examined is, respectively, E13.5, normal, n = 142, reeler n = 166; E14.5, normal, n = 432,reeler, n = 370. Statistical analysis: *p < 0.05; **p < 0.01, according to a χ2 square analysis. Error bars indicate 1 SE to the mean.
Fig. 7.
Fig. 7.
Areal differences in proliferative behavior of cortical neuron precursors in normal and reeler mouse. Developmental changes in leaving fraction index (proportion of silver grains contained in FG neurons related to the total number of silver grains contained in all labeled neurons) in area 6 (A) and area 3 (B). InA and B, each mean percentage is based on the examination of 256–880 autoradiographically labeled neurons observed in 6–10 sections in two to three different animals from the same litter. Developmental changes in numbers of FG neurons under 1 mm2 of cortical surface in area 6 (C) and area 3 (D) of normal and reeler mouse. In C andD, each mean has been determined by the examination of four nonadjacent sections from two to three different animals of the same litter. E, Developmental changes in the cortical generation rate (FG neurons/T) in area 3;F, differences in the leaving fraction index within the entire cortical neuron population for a pulse performed on E17.5 (normal, n = 241 labeled neurons, 6 sections;reeler, n = 445, 6 sections). Error bars indicate 1 SE to the mean. Statistical analysis (in A, B, E, and F): *p < 0.05; **p < 0.01 according to a χ2analysis; (in C, D): *p < 0.05; **p < 0.01 according to a Mann–WhitneyU test.

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