Postnatal changes in the expressions of serotonin 1A, 1B, and 2A receptors in ten brain stem nuclei of the rat: implication for a sensitive period

Abstract

A critical period in respiratory network development occurs in the rat around postnatal days (P) 12-13, when abrupt neurochemical, metabolic, and physiological changes were evident. As serotonin and its receptors are involved in respiratory modulation, and serotonergic abnormality is implicated in sudden infant death syndrome, we hypothesized that 5-HT receptors are significantly downregulated during the critical period. This was documented recently for 5-HT(2A)R in several respiratory nuclei. The present study represents a comprehensive analysis of postnatal development of 5-HT(1A)R and 5-HT(1B)R in 10 brain stem nuclei and 5-HT(2A)R in six nuclei not previously examined. Optical densitometric analysis of immunohistochemically-reacted neurons from P2 to P21 indicated four developmental patterns of expression: (1) Pattern I: a high level of expression at P2-P11, an abrupt and significant reduction at P12, followed by a plateau until P21 (5-HT(1A)R and 5-HT(1B)R in raphé magnus [RM], raphé obscurus [ROb], raphé pallidus [RP], pre-Bötzinger complex [PBC], nucleus ambiguus [Amb], and hypoglossal nucleus [XII; 5-HT(1A)R only]). (2) Pattern II: a high level at P2-P9, a gradual decline from P9 to P12, followed by a plateau until P21 (5-HT(1A)R and 5-HT(1B)R in the retrotrapezoid nucleus (RTN)/parafacial respiratory group (pFRG)). (3) Pattern III: a high level at P2-P11, followed by a gradual decline until P21 (5-HT(1A)R in the ventrolateral subnucleus of solitary tract nucleus [NTS(VL)] and the non-respiratory cuneate nucleus [CN]). (4) Pattern IV: a relatively constant level maintained from P2 to P21 (5-HT(1A)R in the commissural subnucleus of solitary tract nucleus (NTS(COM)); 5-HT(1B)R in XII, NTS(VL), NTS(COM), and CN; and 5-HT(2A)R in RM, ROb, RP, RTN/pFRG, NTS(VL), and NTS(COM)). Thus, a significant reduction in the expression of 5-HT(1A)R, 5-HT(1B)R, and 5-HT(2A)R in multiple respiratory-related nuclei at P12 is consistent with reduced serotonergic transmission during the critical period, thereby rendering the animals less able to respond adequately to ventilatory distress.

Fig. 1

Low magnification photomicrographs of rat brain stem sections at postnatal day (P) 21 reacted for serotonin (5-HT) 1A receptors (5-HT_1AR) (A1–3), 5-HT_1BR (B1–3), and 5-HT_2AR (C1–3). A4, B4, and C4 are control sections processed with non-immune serum instead of the respective primary antibody. Amb, nucleus ambiguus; CN, cuneate nucleus; NTS_COM, commissural subnucleus of the solitary tract nucleus; NTS_VL, ventrolateral subnucleus of the solitary tract nucleus; PBC, pre-Bötzinger complex; RM, nucleus raphé magnus; ROb, nucleus raphé obscurus; RP, nucleus raphé pallidus; RTN, retrotrapezoid nucleus/parafacial respiratory group; XII, hypoglossal nucleus. Scale bar in A1: 200 µm for A1, A3, B1, B3, and C1–3. Scale bar in A2: 250 µm for A2 and B2. Scale bar in A4: 20 µm for A4, B4, and C4.

Fig. 2

5-HT_1AR-ir neurons in the RM (A), ROb (B), RP (C), PBC (D), Amb) (E, and XII (F) at representative postnatal P2 (A1–F1), P7 (A2–F2), P12 (A3–F3), and P21 (A4–F4). The insets in A1–F1 indicate the locations of each nucleus in a diagrammatic cross section of the medulla or pons. Labeling in neurons of all six nuclear groups followed the type I pattern of development, with relatively high levels from P2 (A1–F1) through P7 (A2–F2) to P11 (not shown), but significantly declined at P12 (A3–F3) and remained low thereafter until P21 (A4–F4). Scale bar: 20 µm for all.

Fig. 3

5-HT_1AR-ir neurons in the RTN/pFRG (labeled as RTN for this region in the inset) (A), (NTS_VL (B), NTS_COM (C), and CN (D) at P2 (A1–D1), P7 (A2–D2), P12 (A3–D3), and P21 (A4–D4). The insets in A1-D1 indicate diagrammatically the locations of these four nuclear groups. Labeling of neurons in the RTN/pFRG was relatively high from P2 (A1) through P7 (A2) to P9, then gradually decreased at P10 through P12 (A3), followed by a plateau at relatively low level until P21 (A4). Labeling of neurons in the NTS_VL and CN exhibited a progressive reduction from P2 to P21 (B1–B4 and D1–D4, respectively). On the other hand, immunoreactivity remained quite constant through the first 3 postnatal weeks in the NTS_COM (C1–C4). Scale bar: 20 µm for all.

Fig. 4

Optical densitometric measurements of immunoreactive product for 5-HT_1AR in the cytoplasm of individual neurons in the RM (A), ROb (B), RP (C), PBC (D), Amb (E), XII (F), RTN/pFRG (G), NTS_VL (H) , NTS_COM (I), and CN (J) from P2 to P21. Data points were presented as mean ± SEM. The first six nuclear groups (A–F) showed pattern I trend of development, with significant reduction of labeling at P12 (P < 0.05). The trend for RTN/pFRG (G) was that of pattern II, whereas those for the NTS_VL (H) and CN (J) were of pattern III. In contrast, NTS_COM (I) assumed a pattern IV trend of development (see text for details). ANOVA yielded significant differences in the 5-HT_1AR expression among ages in the RM, ROb, RP, PBC, Amb, XII, and RTN/pFRG (P < 0.01). Tukey’s Studentized test revealed a significant reduction in the 5-HT_1AR expression in the RM, ROb, RP, PBC, Amb, and XII at P12, compared with their adjacent younger age groups (P11). *, P < 0.05 (Tukey’s Studentized test).

Fig. 5

5-HT_1BR-ir neurons in the RM (A), ROb (B), RP (C), PBC (D), Amb (E), and XII (F) at P2 (A1–F1), P7 (A2–F2), P12 (A3–F3), and P21 (A4–F4). The expression of 5-HT_1BR in the first five nuclear groups (A–E) was relatively high from P2 (A1–E1) through P7 (A2–E2) to P11, then precipitously dropped at P12 (A3–E3), followed by some fluctuations until P21 (A4–E4). The labeling in the XII was relatively stable from P2 to P21 (F1–F4), with statistically insignificant fluctuations. Scale bar: 20 µm for all.

Fig. 6

5-HT_1BR-ir neurons in the RTN/pFRG (A; labeled as RTN in the inset), NTS_VL (B), NTS_COM (C), and CN (D) at P2 (A1–D1), P7 (A2–D2), P12 (A3–D3), and P21 (A4–D4). The labeling in the RTN/pFRG exhibited a relatively high level from P2 (A1) through P7 (A2) to P10, then gradually fell from P10 through P12 (A3) to P13, followed by a plateau at a relatively low level until P21 (A4). The labeling in the NTS_VL, NTS_COM, and CN was relatively constant from P2 to P21 (B1–B4, C1–C4, and D1–D4). Scale bar: 20 µm for all.

Fig. 7

Optical densitometric measurements of immunoreactive product for 5-HT_1BR in the cytoplasm of individual neurons in the RM (A), ROb (B), RP (C), PBC (D), Amb (E), XII (F), RTN/pFRG (G), NTS_VL (H) , NTS_COM (I), and CN (J) from P2 to P21. Data points were presented as mean ± SEM. The labeling in the first five nuclear groups (A–E) followed pattern I trend of development, with a significant decrease at P12 (P < 0.05). The trend for the RTN/pFRG (G) was that of pattern II, whereas those for XII (F), NTS_VL (H), NTS_COM (I), and CN (J) were of pattern IV (see text for details). ANOVA revealed significant differences in 5-HT_1BR expression among ages in the RM, ROb, RP, PBC, and Amb (P < 0.01). Tukey’s Studentized test showed a significant reduction in the RM, ROb, RP, PBC, and Amb at P12, compared with their adjacent younger age groups (P11). *, P < 0.05 (Tukey’s Studentized test).

Fig. 8

5-HT_2AR-ir neurons in the RM (A), ROb (B), RP (C), RTN/pFRG (D), NTS_VL (E), and NTS_COM (F) at P2 (A1–F1), P7 (A2–F2), P12 (A3–F3), and P21 (A4–F4). The labeling was relatively constant with statistically insignificant fluctuations from P2 to P21. Scale bar: 20 µm for all.

Fig. 9

Optical densitometric measurements of immunoreactive product for 5-HT_2AR in the cytoplasm of individual neurons in the RM (A), ROb (B), RP (C), RTN/pFRG (D), NTS_VL (E), and NTS_COM (F) from P2 to P21. Data points represented the mean ± SEM. Labeling in all of the nuclear groups followed a pattern IV trend of development during the first 3 postnatal weeks. ANOVA did not reveal any significant differences in 5-HT_2AR expression among ages in any of these nuclear groups.