Birthdate study of GABAergic neurons in the lumbar spinal cord of the glutamic acid decarboxylase 67-green fluorescent protein knock-in mouse

Abstract

Despite the abundance of studies on γ-aminobutyric acid (GABA) ergic neuron distribution in the mouse developing spinal cord, no investigation has been devoted so far to their birthdates. In order to determine the spinal neurogenesis of a specific phenotype, the GABAergic neurons in the spinal cord, we injected bromodeoxyuridine (BrdU) at different developmental stages of the glutamic acid decarboxylase (GAD)67-green fluorescent protein (GFP) knock-in mice. We thus used GFP to mark GABAergic neurons and labeled newly born cells with the S-phase marker BrdU at different embryonic stages. Distribution of GABAergic neurons labeled with BrdU was then studied in spinal cord sections of 60-day-old mice. Our birthdating studies revealed that GABAergic neurogenesis was present at embryonic day 10.5 (E10.5). Since then, the generation of GABAergic neurons significantly increased, and reached a peak at E11.5. Two waves for the co-localization of GABA and BrdU in the spinal cord were seen at E11.5 and E13.5 in the present study. The vast majority of GABAergic neurons were generated before E14.5. Thereafter, GABA-positive neuron generation decreased drastically. The present results showed that the birthdates of GABAergic neurons in each lamina were different. The peaks of GABAergic neurogenesis in lamina II were at E11.5 and E13.5, while in lamina I and III, they were at E13.5 and E12.5, respectively. The present results suggest that the birthdates of GABAergic neurons vary in different lamina and follow a specific temporal sequence. This will provide valuable information for future functional studies.

Keywords: BrdU; GABAergic neuron; birthdate; mouse; spinal cord.

FIGURE 1

Double-labeling of GFP (green) and BrdU (red) in the spinal cord of the GAD₆₇-GFP knock-in mice following injection of BrdU at E10.5 and E11.5. (A,F) Schematic drawings showing the distribution of BrdU-positive cells and double-labeled neurons in the lumbar spinal cord, taken from P60 mice that had received a single pulse of BrdU at E10.5 (A) and E11.5 (F). (A) The schematic drawing represents a single section and it corresponds to the same section illustrated in (B). (F) The schematic drawing represents a single section and it corresponds to the same section illustrated in (D). Red circles correspond to BrdU-positive cells and blue circles correspond to double-labeled neurons. (B) Note the distribution of BrdU-positive cells after injection of BrdU at E10.5. (C) Higher magnification obtained from a different section, in the approximate position indicated by the rectangle in (A). Double-labeled cells for GFP and BrdU were distributed in the ventral horn (arrowheads). (D) Distribution of E11.5-birthdated BrdU-positive nuclei in the spinal cord. (E,G–I) Double-labeled cells for GFP and BrdU were highly concentrated in the spinal dorsal horn after injection of BrdU at E11.5. Double-labeled cells are indicated by white arrowheads. The borders of the spinal cord lamina are depicted as dashed lines. In (A), “d” is dorsal; “v,” ventral; and “cc” indicates the central canal. The same section orientation is maintained in the following figures. Scale bars = 100 μm in (B,D,E) and 10 μm in (B,I) (applies to G–I).

FIGURE 2

Double-labeling of GFP (green) and BrdU (red) in the spinal cord of the GAD₆₇-GFP knock-in mice following injection of BrdU at E12.5 and E13.5. (A,E) Schematic drawings showing the distribution of BrdU-positive cells and double-labeled neurons in the lumbar spinal cord following injection of BrdU at E12.5 (A) and E13.5 (E). (A) The schematic drawing represents a single section and it corresponds to the same section illustrated in (B). (E) The schematic drawing represents a single section and it corresponds to the same section illustrated in (F). Red circles correspond to BrdU-positive cells and blue circles correspond to double-labeled neurons. (B,C) Distribution of the BrdU-labeled cells and double-labeled neurons in the spinal cord after injection of BrdU at E12.5. (D) Magnified image of the rectangle indicated in (C). Note the higher magnification of double-labeled cells for GFP and BrdU in the deep dorsal horn. (F) The distribution of E13.5-birthdated BrdU-positive nuclei was mainly located in the spinal dorsal horn. (G) The inset in (G) illustrates double-labeled cells for GFP and BrdU in the area around the central canal. (H) Distribution of BrdU-positive cells in the spinal cord after injection of BrdU at E16.5. Double-labeled cells are indicated by white arrowheads (D,G). The borders of the spinal cord lamina are depicted as dashed lines. Scale bars = 100 μm in (B,C,F,H) (apply to G). Scale bars = 10 μm in (D) and 20 μm in the inset in (G).

FIGURE 3

Quantitative analysis of BrdU-positive cells (A) and the co-localization of GFP and BrdU (B) in the spinal cord of the P60 GAD₆₇-GFP knock-in mice following injections of BrdU at different developmental stages. The x-axis depicts the embryonic stages in which BrdU was injected, and the y-axis depicts the number of BrdU-positive cells or the percentage of double-labeled cells among all GABAergic neurons, respectively. The horizontal bar indicates the median. Statistical differences were assessed by non-parametric method, namely Kruskal–Wallis test. *P < 0.05 compared with that of E10.5, E14.5, E15.5, E16.5, and E17.5. ^#P < 0.05 compared with that of E10.5, E15.5, E16.5, and E17.5.

FIGURE 4

Laminar distribution of double-labeled cells for GFP and BrdU in the superficial dorsal horn of GAD₆₇-GFP knock-in mice injected with BrdU at E10.5 (A,E), E11.5 (B,F), E12.5 (C,G), and E13.5 (D,H), respectively. Double-labeled cells are indicated by white arrowheads (E–H). (I) Data showing the percentage of double-labeled cells among GABAergic neurons in lamina I, lamina II, and lamina III of spinal cord at different developmental stages. The horizontal bar indicates the median. Statistical differences were assessed by non-parametric method, namely Kruskal–Wallis test. *P < 0.05 compared with that of E10.5 and E11.5. ^#P < 0.05 compared with that of E10.5 and E12.5. ^$P < 0.05 compared with that of E10.5 and E13.5. Borders of lamina I, lamina II, and lamina III are depicted as dashed lines. Scale bars = 50 μm in (D) (applies to A–D) and 20 μm in (H) (applies to E–H).