Postnatal development of N-methyl-D-aspartate receptor subunits 2A, 2B, 2C, 2D, and 3B immunoreactivity in brain stem respiratory nuclei of the rat

Abstract

Previously, we reported that a critical period in respiratory network development exists in rats around postnatal days (P; P12-P13), when abrupt neurochemical, metabolic, and physiological changes occur. Specifically, the expressions of glutamate and N-methyl-d-aspartate (NMDA) receptor (NR) subunit 1 in the pre-Bötzinger complex (PBC), nucleus ambiguus (Amb), hypoglossal nucleus (XII), and ventrolateral subnucleus of solitary tract nucleus (NTS(VL)) were significantly reduced at P12. To test our hypothesis that other NR subunits also undergo postnatal changes, we undertook an in-depth immunohistochemical study of NR2A, 2B, 2C, 2D, and 3B in these four respiratory nuclei in P2-P21 rats, using the non-respiratory cuneate nucleus (CN) as a control. Our results revealed that: (1) NR2A expression increased gradually from P2 to P11, but fell significantly at P12 in all four respiratory nuclei (but not in the CN), followed by a quick rise and a relative plateau until P21; (2) NR2B expression remained relatively constant from P2 to P21 in all five nuclei examined; (3) NR2C expression had an initial rise from P2 to P3, but remained relatively constant thereafter until P21, except for a significant fall at P12 in the PBC; (4) NR2D expression fell significantly from P2 to P3, then plateaued until P12, and declined again until P21; and (5) in contrast to NR2D, NR3B expression rose gradually from P2 to P21. These patterns reflect a dynamic remodeling of NMDA receptor subunit composition during postnatal development, with a distinct reduction of NR2A expression during the critical period (P12), just as NR1 did in various respiratory nuclei. There was also a potential switch between the neonatal NR2D and the more mature NR3B subunit, possibly around the critical period. Thus, during the critical period, NMDA receptors are undergoing greater adjustments that may contribute to attenuated excitatory synaptic transmission in the respiratory network.

Fig. 1

N-methyl-d-aspartate receptors (NR) subunits 2A (NR2A) immunoreactive (-ir) neurons in the pre-Bötzinger complex (PBC) (A), nucleus ambiguus (Amb) (B), hypoglossal nucleus (XII) (C), ventrolateral subnucleus of the solitary tract nucleus (NTS_VL) (D), and cuneate nucleus (CN) (E) at representative postnatal days P2 (A1-E1), P7 (A2-E2), P12 (A3-E3), and P21 (A4-E4). The insets in A1-E1 indicate the locations of each nucleus in a diagrammatic cross section of the medulla. The expression of NR2A-ir in the PBC, Amb, XII, and NTS_VL neurons increased at P7 (compared with that at P2), but was significantly decreased at P12, followed by a plateau or a decline at P21. The NR2A-ir expression in the CN neurons was relatively constant at four ages presented. Scale bar: 20 μm for all.

Fig. 2

Optical densitometric measurements of immunoreactive product for NR2A in the cytoplasm of individual neurons in the PBC (A), Amb (B), XII (C), NTS_VL (D), and CN (E) from P2 to P21. Data points were presented as mean ± SEM. The NR2A-ir expression in the first four nuclei exhibited a gradual increase from P2 or P3 to P11, then a distinct fall at P12, followed by a small rise and a plateau or a gradual decline until P21. The expression in the CN was relatively constant from P2 to P21. ANOVA yielded significant differences in the NR2A expression among ages in all four respiratory nuclei examined (P < 0.01). Tukey’s Studentized test revealed a significant reduction at P12 as compared to P11 (P < 0.01 for the PBC and XII, P < 0.05 for the Amb and NTS_VL). These graphs in Fig. 2 (as well as those in Figs. 4, 6, 8, and 10) show significance only between one age group and its immediately adjacent younger age groups as analyzed by Tukey’s test. Additional significant differences between any two age groups can be found in the text. *, P < 0.05; **, P < 0.01 (Tukey’s Studentized test).

Fig. 3

NR2B-ir neurons in the PBC (A), Amb (B), XII (C), NTS_VL (D), and CN (E) at P2 (A1-E1), P7 (A2-E2), P12 (A3-E3), and P21 (A4-E4). The insets in A1-E1 indicate the locations of each nucleus in a diagrammatic cross section of the medulla. The NR2B-ir expression in all five nuclei was relatively constant at the four ages shown. Scale bar: 20 μm for all.

Fig. 4

Optical densitometric measurements of immunoreactive product for NR2B in the cytoplasm of individual neurons in the PBC (A), Amb (B), XII (C), NTS_VL (D), and CN (E) from P2 to P21. Data points were presented as mean ± SEM. The expression of NR2B-ir in neurons of all five nuclei was relatively stable during the first three postnatal weeks, with only minor fluctuations throughout. ANOVA failed to reveal any significant differences among the ages in all five nuclei examined (P > 0.01).

Fig. 5

NR2C-ir neurons in the PBC (A), Amb (B), XII (C), NTS_VL (D), and CN (E) at P2 (A1-E1), P7 (A2-E2), P12 (A3-E3), and P21 (A4-E4). The insets in A1-E1 indicate the locations of each nucleus in a diagrammatic cross section of the medulla. In all five nuclei, NR2C-ir expression showed the lowest level at P2, a rise at P7, followed by a plateau at P12 and P21, with a noted exception in the PBC, which had a significant reduction at P12. Scale bar: 20 μm for all.

Fig. 6

Optical densitometric measurements of immunoreactive product for NR2C in the cytoplasm of individual neurons in the PBC (A), Amb (B), XII (C), NTS_VL (D), and CN (E) from P2 to P21. Data points were presented as mean ± SEM. The NR2C-ir expression was the lowest at P2, increased with or without statistical significance at P3, followed by a plateau until P21. A distinct exception was found in the PBC, which showed a significant reduction at P12 (P < 0.05). ANOVA revealed significant differences (P < 0.01) in the NR2C-ir expression among the ages in the PBC and XII, and Tukey’s test indicated a significant rise at P3 for the PBC and XII (P < 0.05), and significant reduction at P12 for the PBC (P < 0.05). *, P < 0.05 (Tukey’s Studentized test).

Fig. 7

NR2D-ir neurons in the PBC (A), Amb (B), XII (C), NTS_VL (D), and CN (E) at P2 (A1-E1), P7 (A2-E2), P12 (A3-E3), and P21 (A4-E4). The insets in A1-E1 indicate the locations of each nucleus in a diagrammatic cross section of the medulla. In all five nuclei, NR2D-ir expression demonstrated the highest level at P2, declining at P7, followed by a plateau at P12, and decreased again at P21, except for NTS_VL in which the expression at P21 was comparable to that at P12. Scale bar: 20 μm for all.

Fig. 8

Optical densitometric measurements of immunoreactive product for NR2D in the cytoplasm of individual neurons in the PBC (A), Amb (B), XII (C), NTS_VL (D), and CN (E) from P2 to P21. Data points were presented as mean ± SEM. NR2D-ir exhibited the highest levels at P2, significantly reduced at P3 for the four respiratory related nuclei, followed by a plateau until P12, then declined again until P17-21. ANOVA revealed significant differences (P < 0.01) among the ages in all five nuclei, and Tukey’s test indicated a significant reduction in the expression at P3 for the four nuclei (P < 0.05 for the PBC and Amb, P < 0.01 for the XII, and P < 0.001 for the NTS_VL), and another significant reduction at P17 for the XII (P <0.01) and CN (P < 0.05). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Tukey’s test).

Fig. 9

NR3B-ir neurons in the PBC (A), Amb (B), XII (C), NTS_VL (D), and CN (E) at P2 (A1-E1), P7 (A2-E2), P12 (A3-E3), and P21 (A4-E4). The insets in A1-E1 indicate the locations of each nucleus in a diagrammatic cross section of the medulla. In the PBC, Amb, XII, and NTS_VL, the expression of NR3B-ir was lowest at P2, increased at P7, followed by a further rise (PBC and Amb) or a plateau (XII and NTS_VL) at P12 and P21. In the CN, however, the expression remained relatively constant during development. Scale bar: 20 μm for all.

Fig. 10

Optical densitometric measurements of immunoreactive product for NR3B in the cytoplasm of individual neurons in the PBC (A), Amb (B), XII (C), NTS_VL (D), and CN (E) from P2 to P21. Data points were presented as mean ± SEM. In the PBC, Amb, and XII, the NR3B-ir exhibited a statistically insignificant dip at P3, followed by a gradual increase until P21, whereas in the NTS_VL, the rise from P2 to P4, or from P2 to P10, was gradual and it was followed by a plateau until P21. The XII and NTS_VL had another statistically insignificant reduction at P11-12. In the CN, NR3B-ir was relatively constant from P2 to P21, with a gentle rise mainly between P2 and P10, after which it plateaued until P21. ANOVA yielded significant differences (P < 0.01) among the ages in the PBC, Amb and XII, but Tukey’s Studentized range test that compared one age group with its adjacent younger age group failed to reveal statistical significance in any comparison between paired adjacent age groups (P > 0.05).